Erb-2 receptor targeting peptide

ABSTRACT

The ErbB-2 receptor, a member of the tyrosine kinase type 1 family of receptors, has been implicated in many human malignancies. Bacteriophage display technology was employed to identify peptides that bound to the extracellular domain of human ErbB-2. The peptide KCCYSL, most frequently occurring in the affinity selected population, was chemically synthesized and characterized for its binding activities to the recombinant extracellular domain of ErbB-2. The synthetic peptide exhibits high specificity to ErbB-2 as well as to ErbB-1, another member of ErbB family. Thus, this peptide can be employed in cancer imaging or therapeutic agent targeting to malignant cells ErbB-2 receptor.

This application claims priority to PCT/us2003/021150, filed on Jul. 3,2003, which claims priority to U.S. Provisional Application No.60/394,174 filed Jul. 3, 2002, the entire contents of which are herebyincorporated by reference.

The government has rights in the present invention pursuant to fundingfrom the Department of the Army, Grant No. DAMD17-00-0351 and Grant No.DAMD17-97-1-7198.

BACKGROUND OF THE INVENTION

The present invention claims priority to application U.S. ProvisionalPatent Application Serial No. 60/394,174 filed on Jul. 3, 2002. Theentire text of the above-referenced disclosure is specificallyincorporated herein by reference without disclaimer.

A. Field of the Invention

The invention pertains to the fields of oncology, protein structure andfunction, and molecular biology. More particularly, the inventionrelates to the identification of ErbB-2 binding peptides and their usein cancer diagnosis and therapy.

B. Related Art

The erbB-2 proto-oncogene, also known as HER-2 or neu, is frequentlyaltered in human cancers (Hynes and Stem, 1994). ErbB-2, along withthree other known homologous proteins, ErbB-1 (epidermal growth factorreceptor, EGFR), ErbB-3, and ErbB-4, form the ErbB family or subclass Iof receptor tyrosine kinases (RTK)(McInnes and Sykes, 1997). Theactivation of ErbB receptors leads to stimulation of cell growth anddivision. Signal transduction in this class of receptor proteins isinitiated through the binding of a growth factor to the extracellulardomain of the receptor, followed by receptor homo- orheterodimerization, activation of intracellular kinase domain, andtyrosine autophosphorylation (McInnes and Sykes, 1997).

The ligands for the ErbB growth factor receptor tyrosine kinase familyare numerous yet similar. All of them are structurally homologous andcontain an epidermal growth factor (EGF)-like motif with six cysteinesat highly conserved positions defining three disulfide loops that giverise to the tricyclic nature of these proteins. Despite their receptorspecificity, most of the ErbB ligands are capable of binding severaldifferent receptors. EGF, transforming growth factor α, and betacellulinbind ErbB1 and the ErbB2/ErbB3 heterodimer (Alimandi et al., 1997);neuregulins associate with ErbB-3 and ErbB-4 (Jones et al., 1998); andepiregulin was shown to complex all three receptors, i.e. ErbB-1,ErbB-3, and ErbB-4 (Shelly et al., 1998). No ligand has been found thatbinds directly to ErbB-2.

Gene amplification and overexpression of ErbB-2 is associated withincreased rates of tumor growth and enhanced rates of metastases (Hynesand Stem, 1994). Although ErbB-2 is also expressed at low levels inseveral normal organs and tissues (De Potter et al., 1989), the elevatedlevels of ErbB-2 in many human malignancies and its extracellularaccessibility makes it an attractive target for the development oftumor-specific agents. The ErbB-2 receptor has been targeted by avariety of substances and modalities, including monoclonal antibodies(Weiner et al., 1995), immunoconjugates (Jinno et al. 1996), vaccines(Disis et al., 1999), anti-sense therapy (Wiechen et al., 1995) and genetherapy (Harris et al., 1994). Recently Herceptin™, a humanizedmonoclonal antibody against ErbB-2 (Baselga et al., 1998), was approvedfor the treatment of metastatic breast cancer. Herceptin™ was shown topossess the anti-tumor activity, but it was also found to aggravatedoxorubicin-induced cardiac dysfunction and, possibly, be cardiotoxic onits own (Piccart, 1999). Thus, there remains a need to identify improvedErbB-2-specific reagents for the treatment of ErbB-2 related cancers.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there are providedmethods for targeting an agent to a cell expressing ErbB-2 comprisingbringing the cancer cell into contact with a peptide-agent complex,wherein the peptide comprises the sequence KCCYSL (SEQ ID NO:1). Theagent may be a diagnostic agent, such as a radiolabel, achemilluminescent label, a fluorescent label, a magnetic spin resonancelabel, or a dye. Radiolabels include astatine²¹¹, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²europium, gallium⁶⁷,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m),yttrium⁹⁰, lutetium¹⁷⁷, samarium¹⁵³, holmium¹⁶⁶, bisumth²¹², bisumuth²¹³and actinium²²⁵. Alternatively, the agent may be a therapeutic agent,such as a chemotherapeutic agent, a radiotherapeutic agent, a toxin, acytokine or a nucleic acid construct.

Generally, the peptide will be between 6 and about 100 residues inlength, more particularly between 6 and about 50 residues in length,more particularly between 6 and about 25 residues in length, and moreparticularly between about 6 and 15 residues in length. The cell may bea cancer cell, such as a breast cancer cell or a prostate cancer cell.The complex may further comprise a linking moiety that connects theagent and the peptide, wherein the linking moiety may be linked to thepeptide through the N-terminal amine, the C-terminal carboxyl group, ora side chain. The cell may be located in a subject, such as a human. Thecomplex may thus be delivered locally or regionally to the cell; thecomplex may be delivered systemically as well. The complex may also bedelivered into vasculature of a tumor comprising the cell.

In another embodiment, there are provided methods for diagnosingErbB-2-positive cancer in a subject comprising (a) administering to thesubject a peptide-diagnostic agent complex, wherein the peptidecomprises the sequence KCCYSL; and (b) assessing the amount and/orlocalization in the subject, of the diagnostic agent. The diagnosticagent may be a radiolabel, a chemilluminescent label, a fluorescentlabel, a magnetic spin resonance label, or a dye. Radiolabels includeastatine²¹¹, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷,¹⁵²europium, gallium⁶⁷, iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹,⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur,technicium^(99m), yttrium⁹⁰, lutetium¹⁷⁷, samarium¹⁵³, holmium¹⁶⁶,bisumth²¹², bisumuth²¹³ and actinium²²⁵.

Generally, the peptide will be between 6 and about 100 residues inlength, more particularly between 6 and about 50 residues in length,more particularly between 6 and about 25 residues in length, and moreparticularly between about 6 and 15 residues in length. The subject maysuffer from breast cancer or a prostate cancer. The complex may furthercomprise a linking moiety that connects the agent and the peptide,wherein the linking moiety may be linked to the peptide through theN-terminal amine, the C-terminal carboxyl group, or a side chain. Thecomplex may thus be delivered locally or regionally to the tumor; thecomplex may be delivered systemically as well. The complex may also bedelivered into vasculature of the tumor.

The patient may or may not have been previously diagnosed with cancer.The patient may has previously received a cancer therapy, or may beconcurrently receiving a cancer therapy. The may be patient at anelevated risk for cancer. The assessing may comprise organ or whole bodyimaging, and may further comprise excising a tumor localized by thediagnostic agent.

In yet another embodiment, there are provided methods for treating anErbB-2-positive cancer in a subject in need thereof comprisingadministering to the subject a peptide-therapeutic agent complex,wherein the peptide comprises the sequence KCCYSL. The therapeutic agentmay be a chemotherapeutic agent, a radiotherapeutic agent, a toxin, acytokine or a nucleic acid construct. The radiolabel may be astatine²¹¹,⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²europium,gallium⁶⁷, iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron,³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur,technicium^(99m), yttrium⁹⁰, lutetium¹⁷⁷, samarium¹⁵³, holmium¹⁶⁶,bisumth²¹², bisumuth²¹³ and actinium²²⁵.

Generally, the peptide will be between 6 and about 100 residues inlength, more particularly between 6 and about 50 residues in length,more particularly between 6 and about 25 residues in length, and moreparticularly between about 6 and 15 residues in length. The complex mayfurther comprise a linking moiety that connects the agent and thepeptide, wherein the linking moiety may be linked to the peptide throughthe N-terminal amine, the C-terminal carboxyl group, or a side chain.The cancer may be breast cancer or prostate cancer. The complex may beadministered more than once, may be delivered local or regional to atumor, or may be delivered systemically. The method may further compriseadministering a second distinct cancer therapy, such as radiotherapy,chemotherapy, immunotherapy or surgery.

Also included are methods for rendering an unresectable ErbB-2-positivetumor resectable comprising administering to a subject having the tumora peptide-therapeutic agent complex, wherein the peptide comprises thesequence KCCYSL; methods for treating metastatic ErbB-2-positive cancercomprising administering to a subject in need thereof apeptide-therapeutic agent complex, wherein the peptide comprises thesequence KCCYSL; methods for preventing recurrent ErbB-2-positive cancercomprising administering to a subject having been successfully treatedfor ErbB-2-positive cancer a peptide-therapeutic agent complex, whereinthe peptide comprises the sequence KCCYSL; and methods for treatingmicroscopic residual disease in ErbB-2-positive cancer comprisingadministering to a subject, following tumor resection, apeptide-therapeutic agent complex, wherein the peptide comprises thesequence KCCYSL.

In still yet another embodiment, there is provided a peptide-agentcomplex, wherein the peptide comprises the sequence KCCYSL. The agentmay be a diagnostic agent, as set forth above, or a therapeutic agent,also as set forth above. Generally, the peptide will be between 6 andabout 100 residues in length, more particularly between 6 and about 50residues in length, more particularly between 6 and about 25 residues inlength, and more particularly between about 6 and 15 residues in length.The complex may further comprise a linking moiety that connects theagent and the peptide, wherein the linking moiety may be linked to thepeptide through the N-terminal amine, the C-terminal carboxyl group, ora side chain.

In still yet a further embodiment, there is provided a pharmaceuticalcomposition comprising a peptide-agent complex, wherein the peptidecomprises the sequence KCCYSL. The agent may be a diagnostic agent, asset forth above, or a therapeutic agent, also as set forth above.Generally, the peptide will be between 6 and about 100 residues inlength, more particularly between 6 and about 50 residues in length,more particularly between 6 and about 25 residues in length, and moreparticularly between about 6 and 15 residues in length. The complex mayfurther comprise a linking moiety that connects the agent and thepeptide, wherein the linking moiety may be linked to the peptide throughthe N-terminal amine, the C-terminal carboxyl group, or a side chain.

Also provided are kits comprising peptide-agent complex in a suitablecontainer, wherein the peptide comprises the sequence KCCYSL. Alsoprovided are isolated and purified peptide compositions comprising apeptide comprising the sequence KCCYSL and a linker molecule coupled tothe peptide, wherein the linker comprises a free reactive group.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1B—The ES-MS analysis of synthetic p6.1 peptide (FIG. 1A), andbiotinylated synthetic p6.1 (FIG. 1B). Arrows, major peaks correspondingto molecular mass of the oxidized p6.1 monomer (712), reduced p6.1monomer (714), oxidized biotinylated p6.1 monomer (938), and reducedbiotinylated p6. 1 monomer (940).

FIGS. 2A-2C—Immunoblot analysis of the binding activity of biotinylatedsynthetic p6.1 peptide (100 pM) to the recombinant human ErbB-2-ECD,bovine serum albumin (BSA), bovine asialofetuin (ASF), and human IgG(FIG. 2B). The quantities of immobilized proteins (in ng) are given onthe top. FIG. 2B—Binding profiles of biotinylated p6.1 and biotinylatedcontrol peptide (CP) to immobilized recombinant ErbB-2-ECD (▾-▾ p6.1,+-+ CP), and ErbB-1 (♦-♦ p6.1, x-x CP). FIG. 2C—Fluorescent titration ofthe ErbB-2-ECD with synthetic p6.1 peptide at 0.2 μM protein (▪), 0.4 μMprotein (●), and 0.6 μM protein (▴).

FIG. 3A-3C—Schematic presentation of the EGF primary structure arrangedto demonstrate the formation of CCY motif due to C₁₄-C₃₁ disulfide bond(FIG. 3A). The letter K next to N₃₂ indicates the lysine correspondingto K₇₁ of AR, and K₁₁ of HRG-α and HRG-β. FIG. 3B—Backbone structure ofoxidized form of p6.1 peptide. FIG. 3C—Sequence alignment of the aminoacids 13-32 fragment of EGF with corresponding fragments of severalother ErbB ligands. The amino acid residues participating in formationof KCCY/F motif colored in red. Arrows, indicate a direction of theKCCY/F motif.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As discussed above, overexpression of ErbB-2 is associated withincreased rates of tumor growth and enhanced rates of metastases (Hynesand Stern, 1994). The relatively high levels of ErbB-2 in many humanmalignancies, along with its extracellular accessibility, make it anattractive target for the development of tumor-specific agents.Herceptin™, a humanized monoclonal antibody against ErbB-2 (Baselga etal., 1998), has recently been approved for the treatment of metastaticbreast cancer. Unfortunately, this reagent was found to aggravatedoxorubicin-induced cardiac dysfunction and, possibly, be cardiotoxic onits own (Piccart, 1999).

Peptides, in contrast to large molecules like antibodies, are known toexhibit less toxicity and possess better pharmacokinetic properties suchas higher target-to-background ratios and faster blood clearance(Fischman et al., 1993). Advances in powerful combinatorialtechnologies, such as the use of bacteriophage display libraries, permitthe rapid screening of large peptide libraries for binding to aparticular antigen or receptor. A number of peptides that bind receptormolecules (Doorbar and Winter, 1994), oncoproteins (Renschler et al.,1995), integrins (Murayama et al., 1996), and tumor-associatedcarbohydrates (Peletskaya et al., 1997, Peletskaya et al., 1996) havebeen identified using bacteriophage display libraries. Bacteriophagedisplay technology has also been employed to optimize binding affinitiesof heregulin variants for the ErbB3 receptor (Ballinger et al., 1998)and to study binding interactions of the heregulin-P EGF domain withErbB3 and ErbB4 (Jones et al., 1998). The present inventors, using thisapproach, attempted to identify peptides that specifically and tightlybound the extracellular domain (ECD) of human ErbB-2.

The inventors identified three peptides from a bacteriophage displaylibrary using affinity selection against recombinant ECD of human ErbB-2(ErbB-2-ECD). One of the isolated peptides, KCCYSL (p6.1), represented75% of selected population and exhibited limited linear homology withseveral proteins that potentially could interact with ErbB-1 receptor.Sequence analysis suggested that the p6.1 peptide may also act as amimetic of a CCY/F motif present in the EGF-like domain of all knownErbB ligands. The 6-amino acid p6.1 peptide was chemically synthesizedand examined for its binding affinities and specificities. Results fromthe binding studies showed that the synthetic p6.1 peptide specificallyrecognized the recombinant ErbB-2-ECD, the ErbB-1 receptor protein, andhuman breast and prostate cancer cells overexpressing ErbB-2. Inconclusion, the results demonstrate that the peptide p6.1, identifiedfrom a random peptide phage display library, is capable of specificinteraction with oncogenically activated members of the ErbB growthfactor receptor family. The p6.1 peptide can thus be used as a vehiclefor specific delivery of labels and agents to cancer cells fortherapeutic and diagnostic purposes.

A. ErbB-2 and ErbB-2 Cancers

The c-erbB gene encodes the epidermal growth factor receptor (EGFr) andis highly homologous to the transforming gene of the avianerythroblastosis virus (Downward et al., 1984). The c-erbB gene is amember of the tyrosine-specific protein kinase family to which manyproto-oncogenes belong. The c-erbB gene has recently been found to besimilar, but distinct from, an oncogene referred to variously as ErbB-2,HER-2 or neu oncogene, now known to be intimately involved in thepathogenesis of a number of cancers.

The ErbB-2 gene, which encodes a p185 tumor antigen, was firstidentified in transfection studies in which NIH 3T3 cells weretransfected with DNA from chemically induced rat neuroglioblastomas(Shih et al., 1981). The p185 protein has an extracellular,transmembrane, and intracellular domain, and therefore has a structureconsistent with that of a growth factor receptor (Schechter et al.,1984). The human ErbB-2 gene was first isolated due to its homology withv-erbB and EGF-r probes.

The ErbB-2 oncogene is of particular importance to medical sciencebecause its presence is correlated with the incidence of cancers of thehuman breast and female genital tract. Moreover,amplification/overexpression of this gene has been directly correlatedwith relapse and survival in human breast cancer (Slamon et al., 1987).

B. Peptides and Peptide Conjugates

The present invention focuses on peptides comprising the sequenceKCCYSL, designated p6.1. p6.1 selected from random 6-amino acid phagepeptide library, exhibited specific binding activity to recombinantErbB-2-ECD and to human cancer cells expressing ErbB-2 receptor.Remarkably, the disulfide constrained p6.1 peptide was derived from anunconstrained random peptide library. So far, only a few unconstrainedlibraries have yielded putative disulfide constrained sequences (Devlinet al., 1990, Kay et al., 1993). Most of the reporteddisulfide-containing peptides have been derived from libraries comprisedof constrained peptides by design (Giebel et al., 1995, Oldenburg etal., 1992, Wrighton et al., 1996).

In this study, the inventors used a library with random peptidesinserted near the N-terminus of the mature M13 viral pIII protein. Thereare eight cysteine residues in the mature pIII molecule. Since the phageparticles are maintained in an oxidizing environment, one can reasonablysuggest that two cysteines of the p6.1 insert could be disulfide-bondednot only with each other, but with the cysteines of the pIII protein aswell. The latter, however, would likely interfere with the pIIIstructure resulting in a reduction of phage infectivity andunder-presentation of the clone in the library. Thus, the inventorsbelieve that the cysteine residues of p6.1 displayed on the phagesurface participate in disulfide bond formation within the peptideinsert. Nevertheless, there is a possibility that the insert may existin a reduced linear form on the surface of the phage.

Synthetic p6.1 exhibited binding activity not only to ErbB-2, but alsoto another member of the ErbB receptor family, ErbB-1. This resultsupported that of an overall 40-50% homology between ErbB-2 and ErbB-1.The inventors suggest that the binding activity of p6.1 peptide towardboth ErbB-2 and ErbB-1 is related to its constrained nature. Theoxidized form of p6.1 (FIG. 3B) could mimic a CC(Y/F) motif, found inthe structure of the EGF-like domain of all known ErbB ligands (FIG.3C). The CC(Y/F) motif is formed due to the C₁₄-C₃₁, (EGF numbering).disulfide bridge (FIG. 3A). The residues participating in the formationof this motif, two cysteines and a semi-conservative aromatic amino acid(Y/F), are invariant in all of the ErbB family ligands (FIG. 3C). Suchinvariance suggests the importance of these amino acids in definingeither a structure or the binding properties of the EGF-like proteins.Phenylalanine from the CCF motif of TGF-α was shown to appear on itsbinding interface (McInnes and Sykes, 1997). Remarkably, anotherEGF-like polypeptide, Cripto-1, which is not known to bind any of theErbB-2 receptors, does not contain CCY/F motif in its structure.

Having identified a key structure in ErbB receptor-binding, theinventors also contemplated that variants of the KCCYSL sequence may beemployed. For example, certain non-natural amino acids that satisfy thestructural constraints of the KCCYSL sequence may be substituted withouta loss, and perhaps with an improvement in, biological function, i.e.,ErbB-2 binding.

In addition, the present inventors also contemplate that structurallysimilar compounds may be formulated to mimic the key portions of peptideor polypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Methods for generating specific structures have been disclosed in theart. For example, α-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Methods for generatingconformationally restricted β-turns and β-bulges are described, forexample, in U.S. Pat. Nos. 5,440,013; 5,618,914; and 5,670,155. Othertypes of mimetic turns include reverse and γ-turns. Reverse turnmimetics are disclosed in U.S. Pat. Nos. 5,475,085 and 5,929,237, andγ-turn mimetics are described in U.S. Pat. Nos. 5,672,681 and 5,674,976.

1. Peptide Synthesis

It will be advantageous to produce peptides using the solid-phasesynthetic techniques (Merrifield, 1963). Other peptide synthesistechniques are well known to those of skill in the art (Bodanszky etal., 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia,1984). Appropriate protective groups for use in such syntheses will befound in the above texts, as well as in Protective Groups in OrganicChemistry, 1973. These synthetic methods involve the sequential additionof one or more amino acid residues or suitable protected amino acidresidues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group, such as lysine.

Using solid phase synthesis as an example, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptides of the invention are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

2. Linking Agents

Bifunctional cross-linking reagents have been extensively used for avariety of purposes including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofligand and receptor binding sites, and structural studies.Homobifunctional reagents that carry two identical functional groupsproved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino,sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

Exemplary methods for cross-linking ligands to liposomes are describedin U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511, eachspecifically incorporated herein by reference in its entirety). Variousagents can be covalently bound to peptides through the cross-linking ofamine residues. Liposomes, in particular, multilamellar vesicles (MLV)or unilamellar vesicles such as microemulsified liposomes (MEL) andlarge unilamellar liposomes (LUVET), each containingphosphatidylethanolamine (PE), have linked by established procedures.The inclusion of PE in the liposome provides an active functionalresidue, a primary amine, on the liposomal surface for cross-linkingpurposes. ST peptides are bound covalently to discrete sites on theliposome surfaces. The number and surface density of these sites will bedictated by the liposome formulation and the liposome type. Theliposomal surfaces may also have sites for non-covalent association. Toform covalent conjugates of peptides and liposomes, cross-linkingreagents have been studied for effectiveness and biocompatibility.Cross-linking reagents include glutaraldehyde (GAD), bifunctionaloxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and a watersoluble carbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Through the complex chemistry of cross-linking,linkage of the amine residues of the recognizing substance and liposomesis established.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described in U.S. Pat.No. 5,889,155, specifically incorporated herein by reference in itsentirety. The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups and is thus usefulfor cross-linking polypeptides. Table 1 details certainhetero-bifunctional cross-linkers considered useful in the presentinvention.

TABLE 1 HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length/ LinkerReactive Toward Advantages and Applications after cross-linking SMPTPrimary amines Greater stability 11.2 A Sulfhydryls SPDP Primary aminesThiolation  6.8 A Sulfhydryls Cleavable cross-linking LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Sulfo-LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Water-soluble SMCC Primaryamines Stable maleimide reactive group 11.6 A SulfhydrylsEnzyme-antibody conjugation Hapten-carrier protein conjugationSulfo-SMCC Primary amines Stable maleimide reactive group 11.6 ASulfhydryls Water-soluble Enzyme-antibody conjugation MBS Primary aminesEnzyme-antibody conjugation  9.9 A Sulfhydryls Hapten-carrier proteinconjugation Sulfo-MBS Primary amines Water-soluble  9.9 A SulfhydrylsSIAB Primary amines Enzyme-antibody conjugation 10.6 A SulfhydrylsSulfo-SIAB Primary amines Water-soluble 10.6 A Sulfhydryls SMPB Primaryamines Extended spacer arm 14.5 A Sulfhydryls Enzyme-antibodyconjugation Sulfo-SMPB Primary amines Extended spacer arm 14.5 ASulfhydryls Water-soluble EDC/Sulfo-NHS Primary amines Hapten-Carrierconjugation 0 Carboxyl groups ABH Carbohydrates Reacts with sugar groups11.9 A NonselectiveIn instances where a particular peptide does not contain a residueamenable for a given cross-linking reagent in its native sequence,conservative genetic or synthetic amino acid changes in the primarysequence can be utilized.

3. Diagnostic Agents

In accordance with the present invention, there are provided diagnosticmethods for detecting cancer cells. Many appropriate imaging agents areknown in the art, as are methods for their attachment to antibodies(see, for e.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, eachincorporated herein by reference). The imaging moieties used can beparamagnetic ions; radioactive isotopes; fluorochromes; NMR-detectablesubstances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ⁵¹chromium, ³⁶chlorine,⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²europium, gallium⁶⁷, iodine¹²³,iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶,rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m), bismuth²¹¹,bismuth²¹³ and/or yttrium⁹⁰. Of particular interest are lutetium¹⁷⁷,samarium¹⁵³, holmium¹⁶⁶ and actinium²²⁵. Also, see Table 6, attached.Radioactively labeled ST peptides of the present invention may beproduced according to well-known methods in the art. For instance,monoclonal antibodies can be iodinated by contact with sodium and/orpotassium iodide and a chemical oxidizing agent such as sodiumhypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.ST peptides according to the invention may be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column.Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

4. Therapeutic Agents

The present invention also provides for the delivery of therapeuticagents to cancer cells using ST peptides to target such agents. Theagents may be linked directly to the peptide (above), or they may beencapsulated in a liposome (below) which, in turn, is targeted by the STpeptide. Some examples of therapeutic agents are discussed in thefollowing pages.

a. Radiopharmaceuticals

A number of different radioactive substances can be used in cancertherapy. Examples of radioactive isotopes for therapeutic applicationsinclude astatine²¹¹, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt,copper⁶⁷, ¹⁵²europium, gallium⁶⁷, iodine¹²³, iodine¹²⁵, iodine¹³¹,indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium,³⁵sulphur, technicium^(99m), yttrium⁹⁰, lutetium¹⁷⁷, samarium¹⁵³,holmium¹⁶⁶, bismuth²¹², bismuth²¹³ and actinium²²⁵.

b. Chemopharmaceuticals

The term “chemotherapy” refers to the use of drugs to treat cancer. A“chemotherapeutic agent” is used to connote a compound or compositionthat is administered in the treatment of cancer. One subtype ofchemotherapy known as biochemotherapy involves the combination of achemotherapy with a biological therapy.

Chemotherapeutic agents include, but are not limited to, 5-fluorouracil,bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin(CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin,estrogen receptor binding agents, etoposide (VP16), farnesyl-proteintransferase inhibitors, gemcitabine, ifosfamide, mechlorethamine,melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine,raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC),transplatinum, vinblastine and methotrexate, vincristine, or any analogor derivative variant of the foregoing. These agents or drugs arecategorized by their mode of activity within a cell, for example,whether and at what stage they affect the cell cycle. Alternatively, anagent may be characterized based on its ability to directly cross-linkDNA, to intercalate into DNA, or to induce chromosomal and mitoticaberrations by affecting nucleic acid synthesis. Most chemotherapeuticagents fall into the following categories: alkylating agents,antimetabolites, antitumor antibiotics, corticosteroid hormones, mitoticinhibitors, and nitrosoureas, hormone agents, miscellaneous agents, andany analog or derivative variant thereof

Chemotherapeutic agents and methods of administration, dosages, etc. arewell known to those of skill in the art (see for example, the Goodman &Gilman's “The Pharmacological Basis of Therapeutics” and in “Remington'sPharmaceutical Sciences”, incorporated herein by reference in relevantparts), and may be combined with the invention in light of thedisclosures herein. Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject. Examples of specificchemotherapeutic agents and dose regimes are also described herein. Ofcourse, all of these dosages and agents described herein are exemplaryrather than limiting, and other doses or agents may be used by a skilledartisan for a specific patient or application. Any dosage in-betweenthese points, or range derivable therein is also expected to be of usein the invention.

i. Alkylating agents

Alkylating agents are drugs that directly interact with genomic DNA toprevent the cancer cell from proliferating. This category ofchemotherapeutic drugs represents agents that affect all phases of thecell cycle, that is, they are not phase-specific. Alkylating agents canbe implemented to treat, for example, chronic leukemia, non-Hodgkin'slymphoma, Hodgkin's disease, multiple myeloma, and particular cancers ofthe breast, lung, and ovary. An alkylating agent, may include, but isnot limited to, a nitrogen mustard, an ethylenimene, a methylmelamine,an alkyl sulfonate, a nitrosourea or a triazines.

They include but are not limited to: busulfan, chlorambucil, cisplatin,cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine(mustargen), and melphalan. In specific aspects, troglitazaone can beused to treat cancer in combination with any one or more of thesealkylating agents, some of which are discussed below.

1. Nitrogen Mustards

A nitrogen mustard may be, but is not limited to, mechlorethamine (HN₂),which is used for Hodgkin's disease and non-Hodgkin's lymphomas;cyclophosphamide and/or ifosfamide, which are used in treating suchcancers as acute or chronic lymphocytic leukemias, Hodgkin's disease,non-Hodgkin's lymphomas, multiple myeloma, neuroblastoma, breast, ovary,lung, Wilm's tumor, cervix testis and soft tissue sarcomas; melphalan(L-sarcolysin), which has been used to treat such cancers as multiplemyeloma, breast and ovary; and chlorambucil, which has been used totreat diseases such as, for example, chronic lymphatic (lymphocytic)leukemia, malignant lymphomas including lymphosarcoma, giant follicularlymphoma, Hodgkin's disease and non-Hodgkin's lymphomas.

a. Chlorambucil

Chlorambucil (also known as leukeran) is a bifunctional alkylating agentof the nitrogen mustard type that has been found active against selectedhuman neoplastic diseases. Chlorambucil is known chemically as4-[bis(2-chlorethyl)amino] benzenebutanoic acid.

Chlorambucil is available in tablet form for oral administration. It israpidly and completely absorbed from the gastrointestinal tract. Forexample, after a single oral doses of about 0.6 mg/kg to about 1.2mg/kg, peak plasma chlorambucil levels are reached within one hour andthe terminal half-life of the parent drug is estimated at about 1.5hours. About 0.1 mg/kg/day to about 0.2 mg/kg/day or about 3 6 mg/m²/dayto about 6 mg/m²/day or alternatively about 0.4 mg/kg may be used forantineoplastic treatment. Chlorambucil is not curative by itself but mayproduce clinically useful palliation.

b. Cyclophosphamide

Cyclophosphamide is 2H-1,3,2-Oxazaphosphorin-2-amine,N,N-bis(2-chloroethyl)tetrahydro-, 2-oxide, monohydrate; termed Cytoxanavailable from Mead Johnson; and Neosar available from Adria.Cyclophosphamide is prepared by condensing 3-amino-1-propanol withN,N-bis(2-chlorethyl) phosphoramidic dichloride [(ClCH₂CH₂)2N—POCl₂] indioxane solution under the catalytic influence of triethylamine. Thecondensation is double, involving both the hydroxyl and the aminogroups, thus effecting the cyclization.

Unlike other β-chloroethylamino alkylators, it does not cyclize readilyto the active ethyleneimonium form until activated by hepatic enzymes.Thus, the substance is stable in the gastrointestinal tract, toleratedwell and effective by the oral and parental routes and does not causelocal vesication, necrosis, phlebitis or even pain.

Suitable oral doses for adults include, for example, about 1 mg/kg/dayto about 5 mg/kg/day (usually in combination), depending upongastrointestinal tolerance; or about 1 mg/kg/day to about 2 mg/kg/day,intravenous doses include, for example, initially about 40 mg/kg toabout 50 mg/kg in divided doses over a period of about 2 days to about 5days or about 10 mg/kg to about 15 mg/kg about every 7 days to about 10days or about 3 mg/kg to about 5 mg/kg twice a week or about 1.5mg/kg/day to about 3 mg/kg/day. In some aspects, a dose of about 250mg/kg/day may be administered as an antineoplastic. Because ofgastrointestinal adverse effects, the intravenous route is preferred forloading. During maintenance, a leukocyte count of about 3000/mm³ to4000/mm³ usually is desired. The drug also sometimes is administeredintramuscularly, by infiltration or into body cavities. It is availablein dosage forms for injection of about 100 mg, about 200 mg and about500 mg, and tablets of about 25 mg and about 50 mg.

c. Melphalan

Melphalan, also known as alkeran, L-phenylalanine mustard, phenylalaninemustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative ofnitrogen mustard. Melphalan is a bifunctional alkylating agent which isactive against selective human neoplastic diseases. It is knownchemically as 4-[bis(2-chloroethyl)amino]-L-phenylalanine.

Melphalan is the active L-isomer of the compound and was firstsynthesized in 1953 by Bergel and Stock; the D-isomer, known asmedphalan, is less active against certain animal tumors, and the doseneeded to produce effects on chromosomes is larger than that requiredwith the L-isomer. The racemic (DL-) form is known as merphalan orsarcolysin. Melphalan is insoluble in water and has a pKal of about 2.1.Melphalan is available in tablet form for oral administration and hasbeen used to treat multiple myeloma. Available evidence suggests thatabout one third to one half of the patients with multiple myeloma show afavorable response to oral administration of the drug.

Melphalan has been used in the treatment of epithelial ovariancarcinoma. One commonly employed regimen for the treatment of ovariancarcinoma has been to administer melphalan at a dose of about 0.2 mg/kgdaily for five days as a single course. Courses are repeated about everyfour to five weeks depending upon hematologic tolerance (Smith andRutledge, 1975; Young et al., 1978). Alternatively in certainembodiments, the dose of melphalan used could be as low as about 0.05mg/kg/day or as high as about 3 mg/kg/day or greater.

2. Ethylenimenes and Methymelamines

An ethyleninene and/or a methylmelamine include, but are not limited to,hexamethylmelamine, used to treat ovary cancer; and thiotepa, which hasbeen used to treat bladder, breast and ovary cancer.

3. Alkyl Sulfonates

An alkyl sulfonate includes but is not limited to such drugs asbusulfan, which has been used to treat chronic granulocytic leukemia.

Busulfan (also known as myleran) is a bifunctional alkylating agent.Busulfan is known chemically as 1,4-butanediol dimethanesulfonate.Busulfan is available in tablet form for oral administration, whereinfor example, each scored tablet contains about 2 mg busulfan and theinactive ingredients magnesium stearate and sodium chloride.

Busulfan is indicated for the palliative treatment of chronicmyelogenous (myeloid, myelocytic, granulocytic) leukemia Although notcurative, busulfan reduces the total granulocyte mass, relieves symptomsof the disease, and improves the clinical state of the patient.Approximately 90% of adults with previously untreated chronicmyelogenous leukemia will obtain hematologic remission with regressionor stabilization of organomegaly following the use of busulfan. Busulfanhas been shown to be superior to splenic irradiation with respect tosurvival times and maintenance of hemoglobin levels, and to beequivalent to irradiation at controlling splenomegaly.

4. Nitrosourea

Nitrosureas, like alkylating agents, inhibit DNA repair proteins. Theyare used to treat non-Hodgkin's lymphomas, multiple myeloma, malignantmelanoma, in addition to brain tumors. A nitrosourea include but is notlimited to a carmustine (BCNU), a lomustine (CCNU), a semustine(methyl-CCNU) or a streptozocin. Semustine has been used in such cancersas a primary brain tumor, a stomach or a colon cancer. Stroptozocin hasbeen used to treat diseases such as a malignant pancreatic insulinoma ora malignant carcinoid. Streptozocin has been used to treat such cancersas a malignant melanoma, Hodgkin's disease and soft tissue sarcomas.

a. Carmustine

Carmustine (sterile carmustine) is one of the nitrosoureas used in thetreatment of certain neoplastic diseases. It is 1,3 bis(2-chloroethyl)-1-nitrosourea. It is lyophilized pale yellow flakes orcongealed mass with a molecular weight of 214.06. It is highly solublein alcohol and lipids, and poorly soluble in water. Carmustine isadministered by intravenous infusion after reconstitution as recommended

Although it is generally agreed that carmustine alkylates DNA and RNA,it is not cross resistant with other alkylators. As with othernitrosoureas, it may also inhibit several key enzymatic processes bycarbamoylation of amino acids in proteins.

Carmustine is indicated as palliative therapy as a single agent or inestablished combination therapy with other approved chemotherapeuticagents in brain tumors such as glioblastoma, brainstem glioma,medullobladyoma, astrocytoma, ependymoma, and metastatic brain tumors.Also it has been used in combination with prednisone to treat multiplemyeloma. Carmustine has been used in treating such cancers as a multiplemyeloma or a malignant melanoma. Carmustine has proved useful, in thetreatment of Hodgkin's Disease and in non-Hodgkin's lymphomas, assecondary therapy in combination with other approved drugs in patientswho relapse while being treated with primary therapy, or who fail torespond to primary therapy.

Sterile carmustine is commonly available in 100 mg single dose vials oflyophilized material. The recommended dose of carmustine as a singleagent in previously untreated patients is about 150 mg/m² to about 200mg/m² intravenously every 6 weeks. This may be given as a single dose ordivided into daily injections such as about 75 mg/m² to about 100 mg/m²on 2 successive days. When carmustine is used in combination with othermyelosuppressive drugs or in patients in whom bone marrow reserve isdepleted, the doses should be adjusted accordingly. Doses subsequent tothe initial dose should be adjusted according to the hematologicresponse of the patient to the preceding dose. It is of courseunderstood that other doses may be used in the present invention, forexample about 10 mg/m², about 20 mg/m², about 30 mg/m², about 40 mg/m²,about 50 mg/m², about 60 mg/m², about 70 mg/m², about 80 mg/m², about 90mg/m² to about 100 mg/m².

b. Lomustine

Lomustine is one of the nitrosoureas used in the treatment of certainneoplastic diseases. It is 1-(2-chloro-ethyl)-3-cyclohexyl-1 nitrosoureaIt is a yellow powder with the empirical formula of C₉H₁₆ClN₃O₂ and amolecular weight of 233.71. Lomustine is soluble in 10% ethanol (about0.05 mg/mL) and in absolute alcohol (about 70 mg/mL). Lomustine isrelatively insoluble in water (less than about 0.05 mg/mL). It isrelatively unionized at a physiological pH. Inactive ingredients inlomustine capsules are: magnesium stearate and mannitol.

Although it is generally agreed that lomustine alkylates DNA and RNA, itis not cross resistant with other alkylators. As with othernitrosoureas, it may also inhibit several key enzymatic processes bycarbamoylation of amino acids in proteins.

Lomustine may be given orally. Following oral administration ofradioactive lomustine at doses ranging from about 30 mg/m² to 100 mg/m²,about half of the radioactivity given was excreted in the form ofdegradation products within 24 hours. The serum half-life of themetabolites ranges from about 16 hours to about 2 days. Tissue levelsare comparable to plasma levels at 15 minutes after intravenousadministration.

Lomustine has been shown to be useful as a single agent in addition toother treatment modalities, or in established combination therapy withother approved chemotherapeutic agents in both primary and metastaticbrain tumors, in patients who have already received appropriate surgicaland/or radiotherapeutic procedures. Lomustine has been used to treatsuch cancers as small-cell lung cancer. It has also proved effective insecondary therapy against Hodgkin's Disease in combination with otherapproved drugs in patients who relapse while being treated with primarytherapy, or who fail to respond to primary therapy.

The recommended dose of lomustine in adults and children as a singleagent in previously untreated patients is about 130 mg/m² as a singleoral dose every 6 weeks. In individuals with compromised bone marrowfunction, the dose should be reduced to about 100 mg/m² every 6 weeks.When lomustine is used in combination with other myelosuppressive drugs,the doses should be adjusted accordingly. It is understood that otherdoses may be used for example, about 20 mg/m², about 30 mg/m², about 40mg/m², about 50 mg/m², about 60 mg/m², about 70 mg/m², about 80 mg/m²,about 90 mg/m², about 100 mg/m² to about 120 mg/m².

C. Triazine

A triazine include but is not limited to such drugs as a dacabazine(DTIC; dimethyltriazenoimidaz olecarboxamide), used in the treatment ofsuch cancers as a malignant melanoma, Hodgkin's disease and asoft-tissue sarcoma.

ii. Antimetabolites

Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents,they specifically influence the cell cycle during S phase. They haveused to combat chronic leukemias in addition to tumors of breast, ovaryand the gastrointestinal tract. Antimetabolites can be differentiatedinto various categories, such as folic acid analogs, pyrimidine analogsand purine analogs and related inhibitory compounds. Antimetabolitesinclude but are not limited to, 5-fluorouracil (5-FU), cytarabine(Ara-C), fludarabine, gemcitabine, and methotrexate.

1. Folic Acid Analogs

Folic acid analogs include but are not limited to compounds such asmethotrexate (amethopterin), which has been used in the treatment ofcancers such as acute lymphocytic leukemia, choriocarcinoma, mycosisfungoides, breast, head and neck, lung and osteogenic sarcoma.

2. Pyrimidine Analogs

Pyrimidine analogs include such compounds as cytarabine (cytosinearabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine(fluorode-oxyuridine; FudR). Cytarabine has been used in the treatmentof cancers such as acute granulocytic leukemia and acute lymphocyticleukemias. Floxuridine and 5-fluorouracil have been used in thetreatment of cancers such as breast, colon, stomach, pancreas, ovary,head and neck, urinary bladder and topical premalignant skin lesions.

5-Fluorouracil (5-FU) has the chemical name of5-fluoro-2,4(1H,3H)-pyrimidinedione. Its mechanism of action is thoughtto be by blocking the methylation reaction of deoxyuridylic acid tothymidylic acid. Thus, 5-FU interferes with the synthesis ofdeoxyribonucleic acid (DNA) and to a lesser extent inhibits theformation of ribonucleic acid (RNA). Since DNA and RNA are essential forcell division and proliferation, it is thought that the effect of 5-FUis to create a thymidine deficiency leading to cell death. Thus, theeffect of 5-FU is found in cells that rapidly divide, a characteristicof metastatic cancers.

3. Purine Analogs and Related Inhibitors

Purine analogs and related compounds include, but are not limited to,mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG)and pentostatin (2-deoxycoformycin). Mercaptopurine has been used inacute lymphocytic, acute granulocytic and chronic granulocyticleukemias. Thrioguanine has been used in the treatment of such cancersas acute granulocytic leukemia, acute lymphocytic leukemia and chroniclymphocytic leukemia. Pentostatin has been used in such cancers as hairycell leukemias, mycosis fungoides and chronic lymphocytic leukemia.

c. Natural Products

Natural products generally refer to compounds originally isolated from anatural source, and identified as having a pharmacological activity.Such compounds, analogs and derivatives thereof may be, isolated from anatural source, chemically synthesized or recombinantly produced by anytechnique known to those of skill in the art. Natural products includesuch categories as mitotic inhibitors, antitumor antibiotics, enzymesand biological response modifiers.

i. Mitotic Inhibitors

Mitotic inhibitors include plant alkaloids and other natural agents thatcan inhibit either protein synthesis required for cell division ormitosis. They operate during a specific phase during the cell cycle.Mitotic inhibitors include, for example, docetaxel, etoposide (VP16),teniposide, paclitaxel, taxol, vinblastine, vincristine, andvinorelbine.

1. Epipodophyllotoxins

Epipodophyllotoxins include such compounds as teniposide and VP16. VP16is also known as etoposide and is used primarily for treatment oftesticular tumors, in combination with bleomycin and cisplatin, and incombination with cisplatin for small-cell carcinoma of the lung.Teniposide and VP16 are also active against cancers such as testis,other lung cancer, Hodgkin's disease, non-Hodgkin's lymphomas, acutegranulocytic leukemia, acute non-lymphocytic leukemia, carcinoma of thebreast, and Kaposi's sarcoma associated with acquired immunodeficiencysyndrome (AIDS).

VP16 is available as a solution (e.g., 20 mg/ml) for intravenousadministration and as 50 mg, liquid-filled capsules for oral use. Forsmall-cell carcinoma of the lung, the intravenous dose (in combinationtherapy) can be as much as about 100 mg/m² or as little as about 2mg/m², routinely about 35 mg/m², daily for about 4 days, to about 50mg/m², daily for about 5 days have also been used. When given orally,the dose should be doubled. Hence the doses for small cell lungcarcinoma may be as high as about 200 mg/m² to about 250 mg/m². Theintravenous dose for testicular cancer (in combination therapy) is about50 mg/m² to about 100 mg/m² daily for about 5 days, or about 100 mg/m²on alternate days, for three doses. Cycles of therapy are usuallyrepeated about every 3 to 4 weeks. The drug should be administeredslowly (e.g. about 30 minutes to about 60 minutes) as an infusion inorder to avoid hypotension and bronchospasm, which are probably due tothe solvents used in the formulation.

2. Taxoids

Taxoids are a class of related compounds isolated from the bark of theash tree, Taxus brevifolia. Taxoids include but are not limited tocompounds such as docetaxel and paclitaxel.

Paclitaxel binds to tubulin (at a site distinct from that used by thevinca alkaloids) and promotes the assembly of microtubules. Paclitaxelis being evaluated clinically; it has activity against malignantmelanoma and carcinoma of the ovary. In certain aspects, maximal dosesare about 30 mg/m² per day for about 5 days or about 210 mg/mm² to about250 mg/m² given once about every 3 weeks.

3. Vinca Alkaloids

Vinca alkaloids are a type of plant alkaloid identified to havepharmaceutical activity. They include such compounds as vinblastine(VLB) and vincristine. Vinblastine is an example of a plant alkaloidthat can be used for the treatment of cancer and precancer. When cellsare incubated with vinblastine, dissolution of the microtubules occurs.

Unpredictable absorption has been reported after oral administration ofvinblastine or vincristine. At the usual clinical doses the peakconcentration of each drug in plasma is approximately 0.4 mM.Vinblastine and vincristine bind to plasma proteins. They areextensively concentrated in platelets and to a lesser extent inleukocytes and erythrocytes.

After intravenous injection, vinblastine has a multiphasic pattern ofclearance from the plasma; after distribution, drug disappears fromplasma with half-lives of approximately 1 and 20 hours. Vinblastine ismetabolized in the liver to biologically activate derivativedesacetylvinblastine. Approximately 15% of an administered dose isdetected intact in the urine, and about 10% is recovered in the fecesafter biliary excretion. Doses should be reduced in patients withhepatic dysfunction. At least a 50% reduction in dosage is indicated ifthe concentration of bilirubin in plasma is greater than 3 mg/dl (about50 mM).

Vinblastine sulfate is available in preparations for injection. When thedrug is given intravenously; special precautions must be taken againstsubcutaneous extravasation, since this may cause painful irritation andulceration. The drug should not be injected into an extremity withimpaired circulation. After a single dose of 0.3 mg/kg of body weight,myelosuppression reaches its maximum in about 7 days to about 10 days.If a moderate level of leukopenia (approximately 3000 cells/mm³) is notattained, the weekly dose may be increased gradually by increments ofabout 0.05 mg/kg of body weight. In regimens designed to cure testicularcancer, vinblastine is used in doses of about 0.3 mg/kg about every 3weeks irrespective of blood cell counts or toxicity.

An important clinical use of vinblastine is with bleomycin and cisplatinin the curative therapy of metastatic testicular tumors. Beneficialresponses have been reported in various lymphomas, particularlyHodgkin's disease, where significant improvement may be noted in 50 to90% of cases. The effectiveness of vinblastine in a high proportion oflymphomas is not diminished when the disease is refractory to alkylatingagents. It is also active in Kaposi's sarcoma, testis cancer,neuroblastoma, and Letterer-Siwe disease (bistiocytosis X), as well asin carcinoma of the breast and choriocarcinoma in women.

Doses of about 0.1 mg/kg to about 0.3 mg/kg can be administered or about1.5 mg/m² to about 2 mg/m² can also be administered. Alternatively,about 0.1 mg/m², about 0.12 mg/m², about 0.14 mg/m², about 0.15 mg/m²,about 0.2 mg/m², about 0.25 mg/m², about 0.5 mg/m², about 1.0 mg/m²,about 1.2 mg/m², about 1.4 mg/m², about 1.5 mg/m², about 2.0 mg/m²,about 2.5 mg/m², about 5.0 mg/m², about 6 mg/m², about 8 mg/m², about 9mg/m², about 10 mg/m², to about 20 mg/m², can be given.

Vincristine blocks mitosis and produces metaphase arrest. It seemslikely that most of the biological activities of this drug can beexplained by its ability to bind specifically to tubulin and to blockthe ability of protein to polymerize into microtubules. Throughdisruption of the microtubules of the mitotic apparatus, cell divisionis arrested in metaphase. The inability to segregate chromosomescorrectly during mitosis presumably leads to cell death.

The relatively low toxicity of vincristine for normal marrow cells andepithelial cells make this agent unusual among anti-neoplastic drugs,and it is often included in combination with other myelosuppressiveagents.

Unpredictable absorption has been reported after oral administration ofvinblastine or vincristine. At the usual clinical doses the peakconcentration of each drug in plasma is about 0.4 mM.

Vinblastine and vincristine bind to plasma proteins. They areextensively concentrated in platelets and to a lesser extent inleukocytes and erythrocytes. Vincristine has a multiphasic pattern ofclearance from the plasma; the terminal half-life is about 24 hours. Thedrug is metabolized in the liver, but no biologically active derivativeshave been identified. Doses should be reduced in patients with hepaticdysfunction. At least a 50% reduction in dosage is indicated if theconcentration of bilirubin in plasma is greater than about 3 mg/dl(about 50 mM).

Vincristine sulfate is available as a solution (e.g., 1 mg/ml) forintravenous injection. Vincristine used together with corticosteroids ispresently the treatment of choice to induce remissions in childhoodleukemia; the optimal dosages for these drugs appear to be vincristine,intravenously, about 2 mg/m² of body-surface area, weekly; andprednisone, orally, about 40 mg/m², daily. Adult patients with Hodgkin'sdisease or non-Hodgkin's lymphomas usually receive vincristine as a partof a complex protocol. When used in the MOPP regimen, the recommendeddose of vincristine is about 1.4 mg/m². High doses of vincristine seemto be tolerated better by children with leukemia than by adults, who mayexperience sever neurological toxicity. Administration of the drug morefrequently than every 7 days or at higher doses seems to increase thetoxic manifestations without proportional improvement in the responserate. Precautions should also be used to avoid extravasation duringintravenous administration of vincristine. Vincristine (and vinblastine)can be infused into the arterial blood supply of tumors in doses severaltimes larger than those that can be administered intravenously withcomparable toxicity.

Vincristine has been effective in Hodgkin's disease and other lymphomas.Although it appears to be somewhat less beneficial than vinblastine whenused alone in Hodgkin's disease, when used with mechlorethamine,prednisone, and procarbazine (the so-called MOPP regimen), it is thepreferred treatment for the advanced stages (III and IV) of thisdisease. In non-Hodgkin's lymphomas, vincristine is an important agent,particularly when used with cyclophosphamide, bleomycin, doxorubicin,and prednisone. Vincristine is more useful than vinblastine inlymphocytic leukemia. Beneficial response have been reported in patientswith a variety of other neoplasms, particularly Wilms' tumor,neuroblastoma, brain tumors, rhabdomyosarcoma, small cell lung, andcarcinomas of the breast, bladder, and the male and female reproductivesystems.

Doses of vincristine include about 0.01 mg/kg to about 0.03 mg/kg orabout 0.4 mg/m² to about 1.4 mg/m² can be administered or about 1.5mg/m² to about 2 mg/m² can also be administered. Alternatively, incertain embodiments, about 0.02 mg/m², about 0.05 mg/m², about 0.06mg/m², about 0.07 mg/m², about 0.08 mg/m², about 0.1 mg/m², about 0.12mg/m², about 0.14 mg/m², about 0.15 mg/m², about 0.2 mg/m², about 0.25mg/m² can be given as a constant intravenous infusion.

ii. Antitumor Antibiotics

Antitumor antibiotics have both antimicrobial and cytotoxic activity.These drugs also interfere with DNA by chemically inhibiting enzymes andmitosis or altering cellular membranes. These agents are not phasespecific so they work in all phases of the cell cycle. Thus, they arewidely used for a variety of cancers. Examples of antitumor antibioticsinclude, but are not limited to, bleomycin, dactinomycin, daunorubicin,doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin.Widely used in clinical setting for the treatment of neoplasms thesecompounds generally are administered through intravenous bolusinjections or orally.

Doxorubicin hydrochloride, 5,12-Naphthacenedione,(8s-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-hydrochloride(hydroxydaunorubicin hydrochloride, Adriamycin) is used in a wideantineoplastic spectrum. It binds to DNA and inhibits nucleic acidsynthesis, inhibits mitosis and promotes chromosomal aberrations.

Administered alone, it is the drug of first choice for the treatment ofthyroid adenoma and primary hepatocellular carcinoma. It is a componentof 31 first-choice combinations for the treatment of diseases includingovarian, endometrial and breast tumors, bronchogenic oat-cell carcinoma,non-small cell lung carcinoma, stomach, genitourinary, thyroid, gastricadenocarcinoma, retinoblastoma, neuroblastoma, mycosis fungoides,pancreatic carcinoma, prostatic carcinoma, bladder carcinoma, myeloma,diffuse histiocytic lymphoma, Wilms' tumor, Hodgkin's disease, adrenaltumors, osteogenic sarcoma, soft tissue sarcoma, Ewing's sarcoma,rhabdomyosarcoma and acute lymphocytic leukemia. It is an alternativedrug for the treatment of other diseases such as islet cell, cervical,testicular and adrenocortical cancers. It is also an immunosuppressant.

Doxorubicin is absorbed poorly and is preferably administeredintravenously. The pharmacokinetics are multicompartmental. Distributionphases have half-lives of 12 minutes and 3.3 hours. The eliminationhalf-life is about 30 hours, with about 40% to about 50% secreted intothe bile. Most of the remainder is metabolized in the liver, partly toan active metabolite (doxorubicinol), but a few percent is excreted intothe urine. In the presence of liver impairment, the dose should bereduced.

In certain embodiments, appropriate intravenous doses are, adult, about60 mg/m² to about 75 mg/m² at about 21-day intervals or about 25 mg/m²to about 30 mg/m² on each of 2 or 3 successive days repeated at about 3week to about 4 week intervals or about 20 mg/m² once a week. The lowestdose should be used in elderly patients, when there is prior bone-marrowdepression caused by prior chemotherapy or neoplastic marrow invasion,or when the drug is combined with other myelopoietic suppressant drugs.The dose should be reduced by about 50% if the serum bilirubin liesbetween about 1.2 mg/dL and about 3 mg/dL and by about 75% if aboveabout 3 mg/dL. The lifetime total dose should not exceed about 550 mg/m²in patients with normal heart function and about 400 mg/m² in personshaving received mediastinal irradiation. In certain embodiments, andalternative dose regiment may comprise about 30 mg/m² on each of 3consecutive days, repeated about every 4 week. Exemplary doses may beabout 10 mg/m², about 20 mg/m², about 30 mg/m², about 50 mg/m², about100 mg/m², about 150 mg/m², about 175 mg/m², about 200 mg/m², about 225mg/m², about 250 mg/m², about 275 mg/m², about 300 mg/m², about 350mg/m², about 400 mg/m², about 425 mg/m², about 450 mg/m², about 475mg/m², to about 500 mg/m².

Daunorubicin hydrochloride, 5,12-Naphthacenedione,(8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-lyxo-hexanopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxy-,hydrochloride; also termed cerubidine and available from Wyeth.Daunorubicin (daunomycin; rubidomycin) intercalates into DNA, blocksDAN-directed RNA polymerase and inhibits DNA synthesis. It can preventcell division in doses that do not interfere with nucleic acidsynthesis.

In combination with other drugs it is often included in the first-choicechemotherapy of diseases such as, for example, acute granulocyticleukemia, acute myelocytic leukemia in adults (for induction ofremission), acute lymphocytic leukemia and the acute phase of chronicmyelocytic leukemia. Oral absorption is poor, and it preferably given byother methods (e.g., intravenously). The half-life of distribution is 45minutes and of elimination, about 19 hours. The half-life of its activemetabolite, daunorubicinol, is about 27 hours. Daunorubicin ismetabolized mostly in the liver and also secreted into the bile (about40%). Dosage must be reduced in liver or renal insufficiencies.

Generally, suitable intravenous doses are (base equivalent): adult,younger than 60 years, about 45 mg/m²/day (about 30 mg/m² for patientsolder than 60 year) for about 1 day, about 2 days or about 3 days aboutevery 3 weeks or 4 weeks; or about 0.8 mg/kg/day for about 3 days, about4 days, about 5 days to about 6 days about every 3 weeks or about 4weeks; no more than about 550 mg/m² should be given in a lifetime,except only about 450 mg/m² if there has been chest irradiation;children, about 25 mg/m² once a week unless the age is less than 2 yearsor the body surface less than about 0.5 m, in which case theweight-based adult schedule is used. It is available in injectabledosage forms (base equivalent) of about 20 mg (as the base equivalent toabout 21.4 mg of the hydrochloride). Exemplary doses may be about 10mg/m², about 20 mg/m², about 30 mg/m², about 50 mg/m², about 100 mg/m²,about 150 mg/m², about 175 mg/m², about 200 mg/m², about 225 mg/m²,about 250 mg/m², about 275 mg/m², about 300 mg/m², about 350 mg/m²,about 400 mg/m², about 425 mg/m², about 450 mg/m², about 475 mg/m², toabout 500 mg/m².

Mitomycin (also known as mutamycin and/or mitomycin-C) is an antibioticisolated from the broth of Streptomyces caespitosus which has been shownto have antitumor activity. The compound is heat stable, has a highmelting point, and is freely soluble in organic solvents.

Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid(DNA). The guanine and cytosine content correlates with the degree ofmitomycin-induced cross-linking. At high concentrations of the drug,cellular RNA and protein synthesis are also suppressed. Mitomycin hasbeen used in tumors such as stomach, cervix, colon, breast, pancreas,bladder and head and neck.

In humans, mitomycin is rapidly cleared from the serum after intravenousadministration. Time required to reduce the serum concentration by about50% after a 30 mg bolus injection is 17 minutes. After injection of 30mg, 20 mg, or 10 mg I.V., the maximal serum concentrations were 2.4mg/mL, 1.7 mg/mL, and 0.52 mg/mL, respectively. Clearance is effectedprimarily by metabolism in the liver, but metabolism occurs in othertissues as well. The rate of clearance is inversely proportional to themaximal serum concentration because, it is thought, of saturation of thedegradative pathways. Approximately 10% of a dose of mitomycin isexcreted unchanged in the urine. Since metabolic pathways are saturatedat relatively low doses, the percent of a dose excreted in urineincreases with increasing dose. In children, excretion of intravenouslyadministered mitomycin is similar.

Actinomycin D (Dactinomycin) [50-76-0]; C₆₂H₈₆N₁₂O₁₆ (1255.43) is anantineoplastic drug that inhibits DNA-dependent RNA polymerase. It isoften a component of first-choice combinations for treatment of diseasessuch as, for example, choriocarcinoma, embryonal rhabdomyosarcoma,testicular tumor, Kaposi's sarcoma and Wilms' tumor. Tumors that fail torespond to systemic treatment sometimes respond to local perfusion.Dactinomycin potentiates radiotherapy. It is a secondary (efferent)immunosuppressive.

In certain specific aspects, actinomycin D is used in combination withagents such as, for example, primary surgery, radiotherapy, and otherdrugs, particularly vincristine and cyclophosphamide. Antineoplasticactivity has also been noted in Ewing's tumor, Kaposi's sarcoma, andsoft-tissue sarcomas. Dactinomycin can be effective in women withadvanced cases of choriocarcinoma. It also produces consistent responsesin combination with chlorambucil and methotrexate in patients withmetastatic testicular carcinomas. A response may sometimes be observedin patients with Hodgkin's disease and non-Hodgkin's lymphomas.Dactinomycin has also been used to inhibit immunological responses,particularly the rejection of renal transplants.

Half of the dose is excreted intact into the bile and 10% into theurine; the half-life is about 36 hours. The drug does not pass theblood-brain barrier. Actinomycin D is supplied as a lyophilized powder(0/5 mg in each vial). The usual daily dose is about 10 mg/kg to about15 mg/kg; this is given intravenously for about 5 days; if nomanifestations of toxicity are encountered, additional courses may begiven at intervals of about 3 weeks to about 4 weeks. Daily injectionsof about 100 mg to about 400 mg have been given to children for about 10days to about 14 days; in other regimens, about 3 mg/kg to about 6mg/kg, for a total of about 125 mg/kg, and weekly maintenance doses ofabout 7.5 mg/kg have been used. Although it is safer to administer thedrug into the tubing of an intravenous infusion, direct intravenousinjections have been given, with the precaution of discarding the needleused to withdraw the drug from the vial in order to avoid subcutaneousreaction. Exemplary doses may be about 100 mg/m², about 150mg/m², about175mg/m², about 200mg/m², about 225mg/m², about 250mg/m², about275mg/m², about 300mg/m², about 350mg/m², about 400mg/m², about 425mg/m², about 450 mg/m², about 475 mg/m², to about 500 mg/m².

Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolatedfrom a strain of Streptomyces verticillus. Although the exact mechanismof action of bleomycin is unknown, available evidence would seem toindicate that the main mode of action is the inhibition of DNA synthesiswith some evidence of lesser inhibition of RNA and protein synthesis.

In mice, high concentrations of bleomycin are found in the skin, lungs,kidneys, peritoneum, and lymphatics. Tumor cells of the skin and lungshave been found to have high concentrations of bleomycin in contrast tothe low concentrations found in hematopoietic tissue. The lowconcentrations of bleomycin found in bone marrow may be related to highlevels of bleomycin degradative enzymes found in that tissue.

In patients with a creatinine clearance of greater than about 35 mL perminute, the serum or plasma terminal elimination half-life of bleomycinis approximately 115 minutes. In patients with a creatinine clearance ofless than about 35 mL per minute, the plasma or serum terminalelimination half-life increases exponentially as the creatinineclearance decreases. In humans, about 60% to about 70% of anadministered dose is recovered in the urine as active bleomycin. Inspecific embodiments, bleomycin may be given by the intramuscular,intravenous, or subcutaneous routes. It is freely soluble in water.Because of the possibility of an anaphylactoid reaction, lymphomapatients should be treated with two units or less for the first twodoses. If no acute reaction occurs, then the regular dosage schedule maybe followed.

In preferred aspects, bleomycin should be considered a palliativetreatment. It has been shown to be useful in the management of thefollowing neoplasms either as a single agent or in proven combinationswith other approved chemotherapeutic agents in squamous cell carcinomasuch as head and neck (including mouth, tongue, tonsil, nasopharynx,oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis,larynx), esophagus, lung and genitourinary tract, Hodgkin's disease,non-Hodgkin's lymphoma, skin, penis, cervix, and vulva. It has also beenused in the treatment of lymphomas and testicular carcinoma.

Improvement of Hodgkin's Disease and testicular tumors is prompt andnoted within 2 weeks. If no improvement is seen by this time,improvement is unlikely. Squamous cell cancers respond more slowly,sometimes requiring as long as 3 weeks before any improvement is noted.

d. Miscellaneous Agents

Some chemotherapy agents do not qualify into the previous categoriesbased on their activities. They include, but are not limited to,platinum coordination complexes, anthracenedione, substituted urea,methyl hydrazine derivative, adrenalcortical suppressant, amsacrine,L-asparaginase, and tretinoin. It is contemplated that they are includedwithin the compositions and methods of the present invention for use incombination therapies.

i. Platinum Coordination Complexes

Platinum coordination complexes include such compounds as carboplatinand cisplatin (cis-DDP). Cisplatin has been widely used to treat cancerssuch as, for example, metastatic testicular or ovarian carcinoma,advanced bladder cancer, head or neck cancer, cervical cancer, lungcancer or other tumors. Cisplatin is not absorbed orally and musttherefore be delivered via other routes, such as for example,intravenous, subcutaneous, intratumoral or intraperitoneal injection.Cisplatin can be used alone or in combination with other agents, withefficacious doses used in clinical applications of about 15 mg/m² toabout 20 mg/m² for 5 days every three weeks for a total of three coursesbeing contemplated in certain embodiments. Doses may be, for example,about 0.50 mg/m², about 1.0 mg/m², about 1.50 mg/m², about 1.75 mg/m²,about 2.0 mg/m², about 3.0 mg/m², about 4.0 mg/m², about 5.0 mg/m², toabout 10 mg/m².

ii. Other Agents

An anthracenedione such as mitoxantrone has been used for treating acutegranulocytic leukemia and breast cancer. A substituted urea such ashydroxyurea has been used in treating chronic granulocytic leukemia,polycythemia vera, thrombocytosis and malignant melanoma. A methylhydrazine derivative such as procarbazine (N-methylhydrazine, MIH) hasbeen used in the treatment of Hodgkin's disease. An adrenocorticalsuppressant such as mitotane has been used to treat adrenal cortexcancer, while aminoglutethimide has been used to treat Hodgkin'sdisease.

e. Toxins

Various toxins are also useful in the treatment of cancers. As part ofthe present invention, toxins such as ricin A-chain (Burbage, 1997),diphtheria toxin A (Massuda et al., 1997; Lidor, 1997), pertussis toxinA subunit, E. coli enterotoxin toxin A subunit, cholera toxin A subunitand Pseudomonas toxin c-terminal are suitable. It has demonstrated thattransfection of a plasmid containing the fusion protein regulatablediphtheria toxin A chain gene was cytotoxic for cancer cells.

f. Gene Therapy Vectors

Tumor cell resistance to agents, such as chemotherapeutic andradiotherapeutic agents, represents a major problem in clinicaloncology. One goal of current cancer research is to find ways to improvethe efficacy of one or more anti-cancer agents by combining such anagent with gene therapy. For example, the herpes simplex-thymidinekinase (HS-tK) gene, when delivered to brain tumors by a retroviralvector system, successfully induced susceptibility to the antiviralagent ganciclovir (Culver et al., 1992). In the context of the presentinvention, it is contemplated that gene therapy could be enhanced byspecific cell targeting afforded by ST peptides, as discussed below.

i. Inducers of Cellular Proliferation

In one embodiment of the present invention, it is contemplated thatantisense mRNA directed to a particular inducer of cellularproliferation is used to prevent expression of the inducer of cellularproliferation. The proteins that induce cellular proliferation furtherfall into various categories dependent on function. The commonality ofall of these proteins is their ability to regulate cellularproliferation.

For example, a form of PDGF, the sis oncogene, is a secreted growthfactor. Oncogenes rarely arise from genes encoding growth factors, andat the present, sis is the only known naturally-occurring oncogenicgrowth factor.

The largest class of oncogenes includes the signal transducing proteins(e.g. Src, Abl and Ras). The protein Src is a cytoplasmicprotein-tyrosine kinase, and its transformation from proto-oncogene tooncogene in some cases, results via mutations at tyrosine residue 527.In contrast, transformation of GTPase protein ras from proto-oncogene tooncogene, in one example, results from a valine to glycine mutation atamino acid 12 in the sequence, reducing ras GTPase activity.

Other proteins such as Jun, Fos and Myc are proteins that directly exerttheir effects on nuclear functions as transcription factors.

ii. Inhibitors of Cellular Proliferation

In certain embodiments, the restoration of the activity of an inhibitorof cellular proliferation through a genetic construct is contemplated.Tumor suppressor oncogenes function to inhibit excessive cellularproliferation. The inactivation of these genes destroys their inhibitoryactivity, resulting in unregulated proliferation. The tumor suppressorsRb, p53, p16 and C-CAM are described below.

High levels of mutant p53 have been found in many cells transformed bychemical carcinogenesis, ultraviolet radiation, and several viruses. Thep53 gene is a frequent target of mutational inactivation in a widevariety of human tumors and is already documented to be the mostfrequently mutated gene in common human cancers. It is mutated in over50% of human NSCLC (Hollstein et al., 1991) and in a wide spectrum ofother tumors.

The p53 gene encodes a 393-amino acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are minute by comparison with transformed cells or tumor tissue

Wild-type p53 is recognized as an important growth regulator in manycell types. Missense mutations are common for the p53 gene and areessential for the transforming ability of the oncogene. A single geneticchange prompted by point mutations can create carcinogenic p53. Unlikeother oncogenes, however, p53 point mutations are known to occur in atleast 30 distinct codons, often creating dominant alleles that produceshifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Weinberg, 1991).

Another inhibitor of cellular proliferation is p16. The majortransitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G₁. The activity of thisenzyme may be to phosphorylate Rb at late G₁. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by-an inhibitorysubunit, the p16^(INK4) has been biochemically characterized as aprotein that specifically binds to and inhibits CDK4, and thus mayregulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,1995). Since the p16^(INK4) protein is a CDK4 inhibitor (Serrano, 1993),deletion of this gene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p16 also is known to regulatethe function of CDK6.

p16^(INK4) belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16^(B), p19, p₂₁ ^(WAF1), and p27^(KIP1). Thep16^(INK4) gene maps to 9p21, a chromosome region frequently deleted inmany tumor types. Homozygous deletions and mutations of the p16^(INK4)gene are frequent in human tumor cell lines. This evidence suggests thatthe p16^(INK4) gene is a tumor suppressor gene. This interpretation hasbeen challenged, however, by the observation that the frequency of thep16^(INK4) gene alterations is much lower in primary uncultured tumorsthan in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994; Okamoto et al., 1994; Noboriet al., 1995; Orlow et al., 1994; Arap et al., 1995). Restoration ofwild-type p16^(INK4) function by transfection with a plasmid expressionvector reduced colony formation by some human cancer cell lines(Okamoto, 1994; Arap, 1995).

Other genes that may be employed according to the present inventioninclude Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL,MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27 fusions,anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu,raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes involved inangiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or theirreceptors) and MCC.

iii. Regulators of Programmed Cell Death

In certain embodiments, it is contemplated that genetic constructs thatstimulate apoptosis will be used to promote the death of diseased orundesired tissue. Apoptosis, or programmed cell death, is an essentialprocess for normal embryonic development, maintaining homeostasis inadult tissues, and suppressing carcinogenesis (Kerr et al., 1972). TheBcl-2 family of proteins and ICE-like proteases have been demonstratedto be important regulators and effectors of apoptosis in other systems.The Bcl-2 protein, discovered in association with follicular lymphoma,plays a prominent role in controlling apoptosis and enhancing cellsurvival in response to diverse apoptotic stimuli (Bakhshi et al., 1985;Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985;Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 proteinnow is recognized to be a member of a family of related proteins, whichcan be categorized as death agonists or death antagonists.

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bcl-2 cell death regulatory proteins whichshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl-2 (e.g. Bcl_(XL), Bcl_(W), Bcl_(S), Mcl-1, A1, Bfl-1) or counteractBcl-2 function and promote cell death (e.g. Bax, Bak, Bik, Bim, Bid,Bad, Harakiri).

g. Immunotherapy

An immunotherapeutic agent generally triggers immune effector cells andmolecules to target and destroy cancer cells. The immune effector maybe, for example, an antibody specific for some marker on the surface ofa tumor cell. The antibody alone may serve as an effector of therapy orit may recruit other cells to actually effect cell killing. Variouseffector cells include cytotoxic T cells and NK cells.

i. Immune Stimulators

A specific aspect of immunotherapy is to use an immune stimulatingmolecule as an agent, or more preferably in conjunction with anotheragent, such as for example, a cytokines such as for example IL2, IL-4,IL-12, GM-CSF, tumor necrosis factor; interferons alpha, beta, andgamma; F42K and other cytokine analogs; a chemokine such as for exampleMIP-1, MIP-1β, MCP-1, RANTES, IL-8; or a growth factor such as forexample FLT3 ligand.

One particular cytokine contemplated for use in the present invention istumor necrosis factor. Tumor necrosis factor (TNF; Cachectin) is aglycoprotein that kills some kinds of cancer cells, activates cytokineproduction, activates macrophages and endothelial cells, promotes theproduction of collagen and collagenases, is an inflammatory mediator andalso a mediator of septic shock, and promotes catabolism, fever andsleep. Some infectious agents cause tumor regression through thestimulation of TNF production. TNF can be quite toxic when used alone ineffective doses, so that the optimal regimens probably will use it inlower doses in combination with other drugs. Its immunosuppressiveactions are potentiated by γ-interferon, so that the combinationpotentially is dangerous. A hybrid of TNF and interferon-α also has beenfound to possess anti-cancer activity.

Another cytokine specifically contemplate is interferon α. Interferon αhas been used in treatment of hairy cell leukemia, Kaposi's sarcoma,melanoma, carcinoid, renal cell cancer, ovary cancer, bladder cancer,non-Hodgkin's lymphomas, mycosis fungoides, multiple myeloma, andchronic granulocytic leukemia.

ii. Active Immunotherapy

In active immunotherapy, an antigenic peptide, polypeptide or protein,or an autologous or allogenic tumor cell composition or “vaccine” isadministered, generally with a distinct bacterial adjuvant (Ravindranath& Morton, 1991). In melanoma immunotherapy, those patients who elicithigh IgM response often survive better than those who elicit no or lowIgM antibodies. IgM antibodies are often transient antibodies and theexception to the rule appears to be anti-ganglioside or anticarbohydrateantibodies.

iii. Adoptive Immunotherapy

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL-2 or transduced with genes for tumor necrosis,and readministered (Rosenberg et al., 1988; 1989). To achieve this, onewould administer to an animal, or human patient, an immunologicallyeffective amount of activated lymphocytes in combination with anadjuvant-incorporated antigenic peptide composition as described herein.The activated lymphocytes will most preferably be the patient's owncells that were earlier isolated from a blood or tumor sample andactivated (or “expanded”) in vitro. This form of immunotherapy hasproduced several cases of regression of melanoma and renal carcinoma,but the percentage of responders were few compared to those who did notrespond.

h. Hormonal Therapy

Hormonal therapy may also be used in conjunction with the presentinvention and in combination with any other cancer therapy or agent(s).The use of hormones may be employed in the treatment of certain cancerssuch as breast, prostate, ovarian, or cervical cancer to lower the levelor block the effects of certain hormones such as testosterone orestrogen. This treatment is often used in combination with at least oneother cancer therapy as a treatment option or to reduce the risk ofmetastases.

i. Adrenocorticosteroids

Corticosteroid hormones are useful in treating some types of cancer(e.g. non-Hodgkin's lymphoma, acute and chronic lymphocytic leukemias,breast cancer, and multiple myeloma). Though these hormones have beenused in the treatment of many non-cancer conditions, they are consideredchemotherapy drugs when they are implemented to kill or slow the growthof cancer cells. Corticosteroid hormones can increase the effectivenessof other chemotherapy agents, and consequently, they are frequently usedin combination treatments. Prednisone and dexamethasone are examples ofcorticosteroid hormones.

ii. Other Hormones and Antagonists

Progestins such as hydroxyprogesterone caproate, medroxyprogesteroneacetate, and megestrol acetate have been used in cancers of theendometrium and breast. Estrogens such as diethylstilbestrol and ethinylestradiol have been used in cancers such as breast and prostate.Anti-estrogens such as tamoxifen have been used in cancers such asbreast. Androgens such as testosterone propionate and fluoxymesteronehave also been used in treating breast cancer. Anti-androgens such asflutamide have been used in the treatment of prostate cancer.Gonadotropin-releasing hormone analogs such as leuprolide have been usedin treating prostate cancer.

C. Combination Therapies

Multidrug resistance (MDR) in cancer cells represents a major problem inclinical. medicine. One goal of current research is to find ways toovercome such resistance. In addition, drug combinations are known toreduce the dosages required, and in some cases, produce synergisticeffects. Thus, in order to increase the effectiveness of peptide-basedtherapies described herein, it may be desirable to combine thesecompositions with other agents effective in the treatment of cancer. Atherapeutic agent is capable of negatively affecting cancer cell growthin a subject, for example, by killing cancer cells, reducing the growthrate of cancer cells, or otherwise increasing the quality of life of theafflicted subject. This process may involve contacting the subject withthe peptide/agent and the second therapy at the same time. This may beachieved by contacting the cell with a single composition orpharmacological formulation that includes both the peptide/agent and thesecond therapy, or by contacting the cell with two distinct compositionsor formulations, at the same time, wherein one composition includes thepeptide/agent and the other includes the second agent.

Alternatively, the peptide/agent therapy may precede or follow thesecond therapy by intervals ranging from minutes to weeks. Inembodiments where the other agent and peptide/agent are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the second agent and peptide/agent would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one may contact the cell with both modalities withinabout 12-24 h of each other and, more preferably, within about 6-12 h ofeach other. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several d (2, 3, 4,5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

Various combinations may be employed, peptide/agent therapy is “A” andthe secondary agent is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/AAdministration of the peptides of the present invention to a patientwill follow general protocols for the administration of antibiotics,taking into account the toxicity, if any. It is expected that thetreatment cycles would be repeated as necessary. Listed below are somecommon antibiotics and their respective dosages.D. Liposomal Delivery

In particular embodiments, the peptides of the present invention may beused in conjunction with lipid delivery vehicles, often calledliposomes. A “liposome” is a generic term encompassing a variety ofsingle and multilamellar lipid vehicles formed by the generation ofenclosed lipid bilayers or aggregates. Liposomes may be characterized ashaving vesicular structures with a bilayer membrane, generallycomprising a phospholipid, and an inner medium that generally comprisesan aqueous composition.

A multilamellar liposome has multiple lipid layers separated by aqueousmedium. They form spontaneously when lipids comprising phospholipids aresuspended in an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Lipophilic molecules or molecules with lipophilicregions may also dissolve in or associate with the lipid bilayer.

A liposome used according to the present invention can be made bydifferent methods, as would be known to one of ordinary skill in theart. For example, a phospholipid (Avanti Polar Lipids, Alabaster, Ala.),such as for example the neutral phospholipid dioleoylphosphatidylcholine(DOPC), is dissolved in tert-butanol. The lipid(s) is then mixed withthe imexon and/or a derivative thereof, and/or other component(s). Tween20 is added to the lipid mixture such that Tween 20 is about 5% of thecomposition's weight. Excess tert-butanol is added to this mixture suchthat the volume of tert-butanol is at least 95%. The mixture isvortexed, frozen in a dry ice/acetone bath and lyophilized overnight.The lyophilized preparation is stored at −20° C. and can be used up tothree months. When required the lyophilized liposomes are reconstitutedin 0.9% saline. The average diameter of the particles obtained usingTween 20 is about 0.7 to about 1.0 μm in diameter.

Alternatively, a liposome can be prepared by mixing lipids in a solventin a container, e.g., a glass, pear-shaped flask. The container shouldhave a volume ten-times greater than the volume of the expectedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The solventnormally is removed within about 5 min. to 2 hours, depending on thedesired volume of the liposomes. The composition can be dried further ina desiccator under vacuum. The dried lipids generally are discardedafter about 1 week because of a tendency to deteriorate with time.

Dried lipids can be hydrated at approximately 25-50 mM phospholipid insterile, pyrogen-free water by shaking until all the lipid film isresuspended. The aqueous liposomes can be then separated into aliquots,each placed in a vial, lyophilized and sealed under vacuum.

In other alternative methods, liposomes can be prepared in accordancewith other known laboratory procedures (e.g., see Bangham et al., 1965;Gregoriadis, 1979; Deamer and Nichols, 1983; Szoka and Papahadjopoulos,1978, each incorporated herein by reference in relevant part). Thesemethods differ in their respective abilities to entrap aqueous materialand their respective aqueous space-to-lipid ratios.

The dried lipids or lyophilized liposomes prepared as described abovemay be dehydrated and reconstituted in a solution of inhibitory peptideand diluted to an appropriate concentration with an suitable solvent,e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer.Unencapsulated additional materials, such as agents including but notlimited to hormones, drugs, nucleic acid constructs and the like, areremoved by centrifugation at 29,000×g and the liposomal pellets washed.The washed liposomes are resuspended at an appropriate totalphospholipid concentration, e.g., about 50-200 mM. The amount ofadditional material or active agent encapsulated can be determined inaccordance with standard methods. After determination of the amount ofadditional material or active agent encapsulated in the liposomepreparation, the liposomes may be diluted to appropriate concentrationsand stored at 4° C. until use. A pharmaceutical composition comprisingthe liposomes will usually include a sterile, pharmaceuticallyacceptable carrier or diluent, such as water or saline solution.

The size of a liposome varies depending on the method of synthesis.Liposomes in the present invention can be a variety of sizes. In certainembodiments, the liposomes are small, e.g., less than about 100 nm,about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less than about50 nm in external diameter. In preparing such liposomes, any protocoldescribed herein, or as would be known to one of ordinary skill in theart may be used. Additional non-limiting examples of preparing liposomesare described in U.S. Pat. Nos. 4,728,578, 4,728,575, 4,737,323,4,533,254, 4,162,282, 4,310,505, and 4,921,706; InternationalApplications PCT/US85/01161 and PCT/US89/05040; U.K. Patent ApplicationGB 2193095 A; Mayer et al., 1986; Mayhew et al., 1984, each incorporatedherein by reference).

A liposome suspended in an aqueous solution is generally in the shape ofa spherical vesicle, having one or more concentric layers of lipidbilayer molecules. Each layer consists of a parallel array of moleculesrepresented by the formula XY, wherein X is a hydrophilic moiety and Yis a hydrophobic moiety. In aqueous suspension, the concentric layersare arranged such that the hydrophilic moieties tend to remain incontact with an aqueous phase and the hydrophobic regions tend toself-associate. For example, when aqueous phases are present both withinand without the liposome, the lipid molecules may form a bilayer, knownas a lamella, of the arrangement XY-YX. Aggregates of lipids may formwhen the hydrophilic and hydrophobic parts of more than one lipidmolecule become associated with each other. The size and shape of theseaggregates will depend upon many different variables, such as the natureof the solvent and the presence of other compounds in the solution.

The production of lipid formulations often is accomplished by sonicationor serial extrusion of liposomal mixtures after (I) reverse phaseevaporation (II) dehydration-rehydration (III) detergent dialysis and(IV) thin film hydration. In one aspect, a contemplated method forpreparing liposomes in certain embodiments is heating sonicating, andsequential extrusion of the lipids through filters or membranes ofdecreasing pore size, thereby resulting in the formation of small,stable liposome structures. This preparation produces liposomes only ofappropriate and uniform size, which are structurally stable and producemaximal activity. Such techniques are well-known to those of skill inthe art (see, for example Martin, 1990).

Numerous disease treatments are using lipid based gene transferstrategies to enhance conventional or establish novel therapies.Advances in liposome formulations have improved the efficiency of genetransfer in vivo (Templeton et al., 1997) and it is contemplated thatliposomes are prepared by these methods. Alternate methods of preparinglipid-based formulations for nucleic acid delivery are described (WO99/18933).

In another liposome formulation, an amphipathic vehicle called a solventdilution microcarrier (SDMC) enables integration of particular moleculesinto the bilayer of the lipid vehicle (U.S. Pat. No. 5,879,703). TheSDMCs can be used to deliver lipopolysaccharides, polypeptides, nucleicacids and the like. Of course, any other methods of liposome preparationcan be used by the skilled artisan to obtain a desired liposomeformulation in the present invention.

E. Therapeutic Uses

1. Formulations and Routes of Administration

Pharmaceutical aqueous compositions of the present invention comprise aneffective amount of a peptide dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, antibacterial and antifungal agents, isotonic andabsorption delaying agents and the like. The use of such media andagents for pharmaceutical active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The compositions will be sterile, be fluid to theextent that easy syringability exists, stable under the conditions ofmanufacture and storage.

The actual dosage amount of a composition of the present inventionadministered to a patient can be determined by physical andphysiological factors such as body weight, severity of condition,idiopathy of the patient and on the route of administration. With theseconsiderations in mind, the dosage of a lipid composition for aparticular subject and/or course of treatment can readily be determined.

Although it is most preferred that compositions of the present inventionbe prepared in sterile water containing other non-active ingredients,made suitable for injection, solutions of such active ingredients canalso be prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose, if desired. Dispersions can also be prepared inliquid polyethylene glycols, and mixtures thereof and in oils. Thecarrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants.

The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

2. Indications

ErbB-2 is overexpressed in a number of cancer types including thoseinvolving the female genital tract (e.g., endometrial cancer), gastriccancer and prostate cancer. A preliminary clinical study using a variantof Herceptin™ showed some improvement in patients with prostate andkidney cancer. However, a primary target for ErbB-2 targeted therapiesis breast cancer, of which 20-30% show overexpression of this marker.Thus, in accordance with the present invention, there are providedtherapeutic methods designed to intervene in such ErbB-2 relatedcancers. These therapies may facilitate tumor growth inhibition,reduction in tumor size, induction of apoptosis in tumor cells,inhibition or reduction in metastasis formation, or otherwise result inan improvement in the clinical situation of a cancer patient, includingimproving one or more symptoms of cancer.

The compositions of the present invention can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, rectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, intravesicularlly, mucosally,intrapericardially, orally, topically, locally using aerosol, injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly or via a catheter or lavage. In particular, the invention mayprovide local, regional or systemic administration with respect to thetumor location. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for preparing solutions or suspensions upon the addition of aliquid prior to injection can also be prepared; and the preparations canalso be emulsified.

F. Kits

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, a peptide or analogue thereof may be comprised ina kit. The kits will thus comprise, in suitable container means, apeptide, with optional additional agents of the present invention, suchas linking reagents or diagnostic or therapeutic agents.

The kits may comprise a suitably aliquoted peptide or analogues thereof,whether conjugated or not. The components of the kits may be packagedeither in aqueous media or in lyophilized form. The container means ofthe kits will generally include at least one vial, test tube, flask,bottle, syringe or other container means, into which a component may beplaced, and preferably, suitably aliquoted. Where there is more than onecomponent in the kit, the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be comprised in a vial. The kits of the present invention also willtypically include a means for containing the containers in closeconfinement for commercial sale. Such means may include injection orblow-molded plastic containers into which the desired vials areretained.

G. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

Cell Culture and Maintenance. The HEK-293 human embryonic kidney cells,as well as DU-145 human prostate carcinoma, and T-24 human bladdercarcinoma cell lines were purchased from ATCC. The MDA-MB-435 humanbreast carcinoma cell line was kindly provided by Dr. Janet E. Price, M.D. Anderson Cancer Center, Houston, Tex. All cells were maintained asmonolayer cultures in either DMEM (HEK-293 human embryonic kidney cells)or RPMI-1640 (DU-145 human prostate carcinoma, MDA-MB435 human breastcarcinoma, and T-24 human bladder carcinoma cells) medium supplementedwith 10% fetal bovine serum (FBS), sodium pyruvate, nonessential aminoacids, and L-glutamine. The cultures were maintained at 37° C. in a 5%CO₂ humidified incubator. Subculturing was performed using standardtrypsinization procedures.

Expression and Purification of ErbB-2-ECD. The eukaryotic expressionplasmid c-erbB-2-pRc/CMV_(FLAG) encoding ErbB-2-ECD tagged with a FLAGepitope at C-terminus was used to create a cell line stable expressingthe FLAG-tagged ErbB2-ECD fusion protein (Clark et al., 1997). The humanembryonic kidney cells HEK-293 were transfected with 20 μg ofrecombinant DNA using a calcium phosphate precipitation kit (Stratagene)in accordance to manufacturer's protocol. Stable transfectants wereselected in medium (DMEM+10% FBS) containing G418 (0.5 mg/ml).Individual clones arising from single cells were isolated using 8×10cloning cylinders (Sigma). The supernatant from each clone was assayedfor the expression of recombinant protein by Western blot and ELISAusing Neu (9G6) anti-ErbB-2 mouse monoclonal antibody (Santa CruzBiotechnology). The highest expressing clone was chosen for futurestudies.

The filtered supernatant taken from HEK293 cells that secretedErbB-2-ECD into the medium, was run on anti-FLAG M2 affinity columnequilibrated with 20 mM Tris (pH=8.0), 150 mM NaCl. Bound protein waseluted with 0.1 M glycine (pH=3.0) and neutralized with 1M Tris HCl(pH=8.0). The protein was concentrated and dialyzed against PBS, andNaN₃ was added to prevent bacterial growth.

Western Blot Analysis of ErbB-2-ECD. Purified ErbB-2-ECD was subjectedto SDS-polyacrylamide gel electrophoresis (Laemmli, 1970), andtransferred to a nitrocellulose filter. The filter was subsequentlyblocked with 1% BSA in PBS (pH=7.4), and exposed to Neu (9G6)anti-ErbB-2 mouse monoclonal antibody. The bound antibody was detectedby anti-mouse secondary antibody coupled to alkaline phosphatase. Colorreaction was performed using 5-bromo-4-chloro-3indonyl phosphate (BCIP)and nitro blue tetrazolium (NBT).

Size-Exclusion HPLC Analysis. Size-exclusion chromatography wasperformed on a BIOPSEP SEC-S2000 column equilibrated with 0.05 Mphosphate buffer pH=6.8. A flow rate of 0.5 ml/min was maintainedthroughout the studies. The elution volume of dextran served as voidvolume. Trypsin inhibitor, chicken egg albumin, human albumin, and mouseIgG served as molecular weight markers. Recombinant ErbB-2-ECD (10 μg)was used to inject the column. The elution volume for each injectedprotein was measured as a function of protein molecular weight.

Affinity Selection. The affinity selection was performed following aprotocol described by Smith and Scott (1993). Briefly, wells of themicrotiter plates were first coated with streptavidin, washed with TPBS(phosphate buffered saline, pH 7.4, 0.5% (v/v) Tween-20), and thenblocked with 3% (w/v) BSA. Biotinylated antigen was incubated withstreptavidin coated plates for 2 hours at 4° C. In first and secondround of selection 10 μg of biotinylated antigen per plate was added, inthird round amount of antigen was decreased to 1 μg, and for fourthround of selection 0.1 μg of antigen was used. Unoccupied biotin-bindingsites were blocked with 0.1 mM biotin. Phage was incubated with antigenfor 4 hrs at RT. Plates were washed 10 times with TPBS to remove unboundphage. Bound phage was eluted with elution buffer (0.1 M HCl, pH isadjusted to 2.2 with glycine, 1 mg/ml BSA, 0.1 mg/ml phenol red) at RTduring 10 min. The increased percentage of bound phage to input phage(yield) was an intrinsic characteristic of successful biopanning. Aftereach round, except the last, the eluate of the bound phage waspropagated and used as input for the next round. Individual clones werecharacterized by DNA sequencing after the fourth round of selection.

DNA sequencing analysis. Individual phage isolates were sequencedmanually by a modified dideoxy sequencing methodology as described byHaas and Smith (1993).

Sequence Comparison. The sequence homology searches were performed usingthe FASTA program of the University of Wisconsin Genetics Computer Groupprogram package (GCG, version 10.0-Unix, January 1999). EMBL andPIR-protein databases were released March 1997 and June 1999respectively.

Peptide Synthesis. The peptides were chemically synthesized on theApplied Biosystems peptide synthesizer 431A using FMOC-based chemistry.

Mass spectrometric analysis. Mass Consortium Corporation, San Diego,performed the mass spectrometric analysis. Commercially availableoxidized form of gluthatione (Sigma # G4501) was used as control forreduction conditions.

Immunoblot analysis of binding activity of the p6.1 peptide. ErbB-2-ECDand three other proteins, bovine serum albumin (BSA, Sigma #A-3912),asialofetuin (AF, Sigma #A-4781), and human IgG (Sigma #I-2511) wereimmobilized on a nitrocellulose membrane at various quantities (6.25-50ng) and then incubated with 1% blocking reagent (Boehringer Mannheim,cat # 1096 176) in phosphate buffered saline (PBS) pH=7.4. After washingthree times with PBS, the membrane was incubated with 100 μMbiotinylated peptide for 2 hrs at RT. The membrane was washed andincubated with alkaline phosphatase labeled streptavidin (Sigma #S-2890)for 1 hr RT. The staining reaction was performed using 50 μl NBT and37.5 μl X-phosphate solutions (Boehringer Mannheim, cat # 1383 213) in10 ml 0.1M Tris buffer, pH=9.5, 0.05 MMgCl, 0.1 M NaCl.

ELISA. 100 ng of ErbB-2-ECD or ErbB1 (Sigma # E-2645) in PBS was appliedto polystyrene wells (Costar #3590) overnight at +4° C. Coated wellswere washed three times with TPBS (phosphate buffered saline, pH 7.4,0.5% (v/v) Tween-20) and blocked with 1% blocking reagent (BoehringerMannheim # 1096 176) in PBS for 1 hr at RT. Wells were subsequentlyincubated with biotinylated peptide added to the wells by serialdilutions (1:2) at RT for 2 hr. After washing three times with TPBS,wells were incubated with alkaline phosphatase labeled streptavidin(Sigma # S-2890) for 1 hr at RT. Bound alkaline phosphatase wasdeveloped using 1 mg/ml p-Nitrophenyl Disodium Phosphate (Sigma #N-9389) in 1M diethanolamine buffer, 0.5 mM MgCl₂, pH=9.8. The pNPPreaction was stopped with 3M NaOH. Optical density was measured at 405nm in a microplate reader. All assays were performed at least threetimes in triplicate wells. Values are reported as an average ofabsorbence +/−SD.

Cell Binding Assay. DU-145 human prostate adenocarcinoma), MDA-MB-435(human breast carcinoma), and T-24 (human bladder carcinoma) cells weregrown directly on the same microscope slide using a 4 well Lab-Tek IIchamber slide system (Nalge Nunc). When cultures reached approximately60-70% confluence, the cells were briefly washed with PBS, fixed with 4%formaldehyde solution in PBS for 30 min but not permeabilized, andpreblocked for 1 hr with 2% bovine serum albumin (BSA) solution in PBSat 37° C. The cultivation chambers were removed and cells were incubatedfor 2 hr with solution of Neu (9G6) anti-ErbB-2 mouse monoclonalantibody (Santa Cruz Biotechnology) diluted 1:100 and biotinylatedpeptide (100 μM) in 2% solution BSA in PBS. The slides were washed threetimes with PBS followed by 1 hr incubation with LissamineRhodamineconjugated donkey anti-mouse IgG (Jackson ImmunoResearchLaboratories) and streptavidin-AMCA-S conjugate (Molecular Probes) in 2%BSA in PBS. After additional washes with PBS, the slides were mountedand analyzed by fluorescent microscopy using Rhodamine and UVA filtersets.

Fluorescence assay. The fluorescence titration studies were carved outusing a SLM Aminco spectrofluorometer interfaced to a DEL 433/L PCrunning SLM Aminco 8100 series 2 software. The titrations were performedat 25° C. with varying amounts of peptide added to a fixed ErbB-2-ECDconcentration (200-600 nM) in 2 ml of phosphate buffered saline(pH=7.4). Each measurement was collected for 5-20 sec after 1-2 minpre-equilibration. The excitation shutter remained closed duringpre-equilibration of the sample and was opened only during dataacquisition in order to minimize photobleaching of the sample. Thefluorescence measurements were corrected for dilutions andphotobleaching of ErbB-2-ECD. Values reported are an average of threeindependent measurements. The difference between total peptideconcentration and free peptide concentration was neglected since theconcentration of the peptide was much higher than concentration of theprotein during the entire experiment. The fitting procedure wasperformed using Origin (Microcal Software Inc).

Example 2 Results

Purification and characterization of the recombinant extracellulardomain of ErbB-2. The ECD of human ErbB-2 tagged with the FLAG epitopeDYKDDDDK (SEQ ID NO:2) was expressed in human embryonic kidney cells(HEK-293) and purified by anti-FLAG affinity chromatography (Clark etal., 1997). The identity of the recombinant protein was confirmed byWestern blot analysis with Neu (9G6) anti-ErbB-2 mouse monoclonalantibody. The purified protein yielded a single species by SDS-PAGEanalysis with molecular weight of approximately 90 kDa. However, sizeexclusion HPLC analysis under non-denaturing conditions revealed themolecular weight of the recombinant ECD two fold higher than SDS-PAGEelectrophoresis ˜182 kDa, suggesting the existence of the protein as ahomodimer in solution. This is consistent with previously reportedobservations describing the ErbB-2-ECD in sera from patients withvarious carcinomas as a homodimer with MW ˜200 kDa (Wu et al., 1993).The affinity purified ErbB-2ECD was biotinylated and used in thebiopanning procedures.

Isolation of peptide sequences that bind ErbB-2 extracellular domain. Arandom 6-amino acid peptide bacteriophage display library was affinityselected against the ErbB-2-ECD to identify novel peptide-ligands. Thelibrary was based on the fUSE5 bacteriophage vector encoding the foreignpeptide insert in N-terminus of minor coat protein pIII (Scott andSmith, 1990). Four rounds of affinity selection were employed to enrichphage populations for virions that displayed peptides with affinity toErbB-2-ECD (Table 1). The analysis of 100 clones from the affinityselected phage population revealed three groups of clones. The largestgroup, which accounted for 75% of the sequenced clones, contained aninsert encoding the KCCYSL peptide (p6.1). Within this group severalnucleotide sequences were found with different bases in the thirdposition of codons but the same amino acid sequence. This supports thefact that the peptide was selected due to peptide-protein interactionand not to propagation properties of the particular clone. The remainingclones fell into two groups with similar sequences WYAWML (SEQ IDNO:3)(p6.2) and WYSWLL (SEQ ID NO:4)(p6.3). Futher investigation of thep6.2 and p6.3 peptides were not pursued due to their low presentation inselected phage population and high hydrophobicity.

TABLE 1 Affinity Selected Peptide Sequences Name Peptide SequenceLibrary % Of Clones* p6.1 KCCYSL F3-6 75 p6.2 WYAWML F3-6 8 p6.3 WYSWLLF3-6 10 *The percent of individual clones that were represented in theaffinity selected and sequenced phage clones.

Homology analysis of selected p6.1 peptide with known protein sequences.The p6.1 peptide was analyzed for stretches of homology with knownprotein sequences from PIR (George et al., 1996) and SPTREMBL (Stoesseret al., 1999) protein sequence databases. The results of the FASTAsequence analysis (Pearson et al., 1988) for p6.1 are summarized inTable 2. The six-amino acid p6.1 peptide is found to share a limitedlinear sequence homology with several different proteins (Table 2),including monocyte chemotactic protein (MCP), whey acidic protein (WAP),human spasrnolytic protein (hSP), and the envelope protein of the Rousassociated virus 1 (RAV 1 Env). These proteins, each containedfive-amino acid stretches of homology to p6.1 with 1 conservativesubstitution (CCYTL (SEQ ID NO:5), KCCFS (SEQ ID NO:6), KCCFS and CCFSL(SEQ ID NO:7) respectively). Of particular interest was the homology ofp6.1 with hSP and RAV 1 Env, since those proteins could be involved indirect interaction with ErbB receptors. Human SP belongs to the trefoilpolypeptide family and has been implicated in the transientphosphorylation of ErbB-1 (Taupin et al., 1999). The homology of p6.1peptide with RAV 1 Env may be indicative of functional significance,since ErbB-1 has been suggested as a possible receptor for severaldifferent viruses (Strong and Lee, 1996; Tang et al, 1993).

TABLE 2 Homology of p6.1 peptide with known protein sequences NameSequence Position p6.1 KCCYSL Monocyte Chemotactic Protein, Bovine CCYTL 9-13** Whey Acidic Protein, Camel KCCFS  97-101** Spasmolytic Protein,Human KCCFS 106-110** Envelope Protein, Rous Associated Virus 1 CCFSL 9-13* ^(a)Bold invariant amino acids; regular, equivalent amino acids.*Amino acid numbers derived from the sequences deposited in STREMBL databank. **Amino acid numbers derived from the sequences deposited in PIRdata bank.

No linear sequence homology to p6.1 was found in any of the ErbB familyligands. However, the oxidized form of p6.1 could mimic a CC(Y/F) motifformed within the EGF-like domain of all known ErbB ligands due toC₁₄-C₃₁ (EGF numbering) disulfide bond. Moreover, three of ErbB ligands,amphiregulin (AR), heregulin-(x (HRG-cc), and heregulin-β1 (HRG-(β1),exhibit four-residue homology (KCCF) due to the presence of lysineresidue next to the cysteine corresponding to C₃₁, of the EGF.

Peptide synthesis and characterization. The p6.1 peptide and itsbiotinylated analog were chemically synthesized and purified to examinetheir binding activity in the absence of phage particle. Electrospraymass spectrometry was employed to confirm the molecular weight of thepeptides and examine their conformations. Since the p6.1 sequencecontained two cysteine residues, the peptide could exist in a reducedform, as an oxidized monomer, as a disulfide-bridged dimer or as amixture. FIG. 1A shows electrospray ionization mass spectrum (ESI-MS) ofp6.1. The major peak corresponds to the ion [M*−H]⁻ at m/z 712 where M*is molecular weight of oxidized form of a single p6.1 molecule. There isalso a peak at m/z 714, which is attributed to the reduced form of p6.1[M−H]⁻. FIG. 1B shows ESI-MS of biotinylated p6.1. There are two majorpeaks from the reduced p6.1 monomer ion [M*−H]⁻ at m/z 940 and from theoxidized p6.1 monomer [M−H]⁻ at m/z 938. These results suggest that thepeptide exists in solution in two forms as an oxidized monomer and areduced monomer. The mass spectrum method did not detect any presence ofdimerized peptide in solution, which was consistent with the HPLCanalysis. Since the molecules become protonated during the ionizationprocedure, reduction conditions of the ESI technique on disulfide bondstability were analyzed. For this purpose a commercially availableoxidized glutathione, that exists as a disulfide-bonded peptide dimer(MW=612) was used. Mass spectrum of glutathione contained two majorpeaks from the ion [M−H]⁻ at m/z 611 and from the [M−2H]²⁻ at m/z 305.There was no peak that corresponded to the reduced form of glutathione.Thus, the ESI procedure did not appear to reduce disulfide bridges andcould be used for detection of the oxidized forms of the peptide.

Binding activity of p6.1 peptide. The binding specificity of syntheticp6.1 peptide to recombinant ErbB-2-ECD was examined by immunoblot andELISA procedures. Immunoblot analysis was performed to compare thebinding activity of the p6.1 peptide to ErbB-2-ECD and several othernon-ErbB-2 related proteins. Purified recombinant ErbB-2-ECD as well asBSA, bovine asialofetuin, and human IgG were spotted on a nitrocellulosemembrane at various amounts and exposed to the biotinylated p6.1peptide. The p6.1 peptide bound only ErbB-2 and did not react withcontrol proteins (FIG. 2A). Further ELISA analysis was performed tocompare: first, binding activities of p6.1 and control peptide toErbB-2ECD and second, the binding activity of p6.1 to ErbB-2-ECD and toErbB-1 receptor, which is a member of the same RTK family. RecombinantErbB-2-ECD and ErbB1 receptor from human carcinoma A431 cells wereapplied to plastic dishes and then incubated with the variousconcentrations of biotinylated p6.1 and a control peptide(biotin-RRLLFYKYVYKRYRAGKQRG (SEQ ID NO:8)). The p6.1 peptidedemonstrated significant dose dependent binding activity to bothErbB-2-ECD and ErbB-1 protein, while the control peptide did not bindeither ErbB-2-ECD or ErbB-1 (FIG. 2B).

Fluorescence quenching studies were performed in order to evaluate theequilibrium dissociation constant of p6.1 to the soluble Erb-2-ECD.Intrinsic tryptophan fluorescence of the protein was monitored as afunction of peptide concentration. Since p6.1 peptide contains atyrosine residue, samples were excited at 300 nm and monitored at 350 nmto minimize the influence of tyrosine emission on tryptophanfluorescence. Titration of ErbB-2-ECD with p6.1 resulted in substantialdecrease of tryptophanyl fluorescence with maximum quenching of 20%under saturation conditions. In the analysis, the inventors assumed amodel with the stoichiometric ratio of protein to peptide equal to 1:1.The data were collected at 2μM, 4 μM and 6 μM concentrations of theprotein. Theoretical curves were generated for the percentage offluorescence quenching as a function of total peptide concentration(FIG. 2C). An apparent equilibrium dissociation constant,K_(d)=30.2×/−7.6 μM, was determined using a nonlinear least-squarescurve fitting procedure, which was applied to all three sets of datapoints obtained at different concentrations of protein with K_(d) as theshared constant for all curves.

Direct Cell Binding Assay. The ability of the p6.1 to recognize culturedhuman cancer cells expressing native conformation of the ErbB-2 wasexamined. High levels of ErbB-2 expression on DU-145 human prostatecarcinoma cells has been reported previously (Zhau et al., 1992). TheMDA-MB-435 human breast carcinoma cells were shown to express moderatelevels of the ErbB-2 protein (Hynes and Stem, 1994). In doubleimmunostaining studies both the anti-ErbB-2 mouse monoclonal antibody,and biotinylated p6.1 peptide bound prostate carcinoma cells DU-145 andMDA-MB-435 cells. This result was consistent with experimental ELISAdata suggesting that the Neu (9G6) antibody used in the studies and thep6.1 peptide do not compete for the same binding site on theextracellular domain of oncoprotein. The biotinylated control peptide(biotin-RRLLFYKYVYKRYRAGKQRG) did not bind DU-145 or MDA-MB-435 cells.Neither the Neu (9G6) anti-ErbB-2 antibody, nor the biotinylated p6.1bound T-24 human bladder carcinoma cells used as negative control.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

H. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for targeting an agent to a cancer cell expressing ErbB-2comprising bringing said cancer cell into contact with a peptide-agentcomplex, wherein said peptide comprises the sequence KCCYSL (SEQ IDNO:1) and said peptide binds to the extracellular domain of ErbB-2,wherein said agent is a diagnostic agent, a chemotherapeutic, aradiotherapeutic, a toxin or a cytokine.
 2. The method of claim 1,wherein said agent is a diagnostic agent.
 3. The method of claim 2,wherein said diagnostic agent is a radiolabel, a chemilluminescentlabel, a fluorescent label, a magnetic spin resonance label, or a dye.4. The method of claim 3, wherein the diagnostic agent is a radiolabelselected from the group consisting of astatine²¹¹ , ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²europium, gallium⁶⁷,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m),yttrium⁹⁰, lutetium¹⁷⁷, samarium¹⁵³, holmium¹⁶⁶, bisumth²¹², bisumuth²¹³and actinium²²⁵,
 5. The method of claim 1, wherein said agent is achemotherapeutic agent.
 6. The method of claim 1, wherein said agent isa radiotherapeutic agent.
 7. The method of claim 1, wherein said peptideis between 6 and about 100 residues in length.
 8. The method of claim 7,wherein said peptide is between 6 and about 50 residues in length. 9.The method of claim 8, wherein said peptide is between 6 and about 25residues in length.
 10. The method of claim 9, wherein said peptide isbetween 6 and about 15 residues in length.
 11. The method of claim 1,wherein said cancer cell is a breast cancer cell.
 12. The method ofclaim 1, wherein said cancer cell is a prostate cancer cell.
 13. Themethod of claim 1, wherein said complex further comprises a linkingmoiety that connects said agent and said peptide.
 14. The method ofclaim 13, wherein said linking moiety is linked to said peptide throughthe N-terminal amine, the C-terminal carboxyl group, or a side chain.15. The method of claim 1, wherein said cancer cell is located in asubject.
 16. The method of claim 15, wherein is said subject is a human.17. The method of claim 15, wherein said complex is delivered local orregional to said cell.
 18. The method of claim 15, wherein said complexis delivered systemically.
 19. The method of claim 1, wherein saidcomplex is delivered into the vasculature of a tumor comprising saidcell.
 20. The method of claim 1, wherein said agent is a toxin.
 21. Themethod of claim 1, wherein said agent is a cytokine.